Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold.Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from  The stereochemistry of the plant heteroyohimbine alkaloids is a key factor determining their diverse biological activities. Here, the authors carry out structural, localization and genetic experiments to understand the mechanism of stereoselectivity for three heteroyohimbine synthases and to identify their function in vivo. Heteroyohimbines are a prevalent subclass of the monoterpene indole alkaloids (Corynanthe type skeleton), having been isolated from many plant species, primarily from the Apocynaceae and Rubiaceae families23489101112141516171819Catharanthus roseus produces three of these isomers, ajmalicine (raubasine), tetrahydroalstonine and 19-epi-ajmalicine (mayumbine)  21. Theseyumbine) 2122. We mbine) 2124. AlthoC. roseus transcriptome25In this study, we assayed 14 MDR homologues identified from the C. roseus transcriptome25C. roseus cDNA and expressed in Escherichia coli, with the exception of Cr_017994, which could not be expressed and was not considered further. The remaining candidates were assayed with the substrate strictosidine aglycone, and product formation was monitored by liquid chromatography mass spectrometry (LC-MS). Of these, four  reduced strictosidine aglycone to a product corresponding to one of the heteroyohimbines  and the predicted active sites have numerous differences . HYS, whsolution ; THAS2, solution ) and in solution ; THAS2, solution ), while solution .The five HYS structures described here are similar to sinapyl alcohol dehydrogenase  and the SAD homologue cinnamyl alcohol dehydrogenase 2728+ in place. Most notably, Glu59 of THAS1 anchors NADP(H) through a bidentate interaction with both ribose hydroxyls, with His59 playing a comparable role in SAD, although here the interaction is with the 3′ OH only  co-substrate binding at the base of the active site . Loop 2, OH only . Glu59 i OH only  and 4b27H only 27. Howeveronly 2727. In THASructures . Closer in situ under aqueous conditions (Methods), 1H,15N-HMBC NMR showed that an enamine species was the predominant product in aqueous methanolic solution  and tetrahydroalstonine (S C20), protonation must also occur from the opposite face to yield R stereochemistry at C20  stereochemistry (kcat (observed) values could be measured for THAS1 (1.518±0.059 s−1), THAS2 (0.033±0.001 s−1), THAS3 (0.102±0.004 s−1), THAS4 (0.044±0.006 s−1), HYS (0.083±0.005 s−1), HYS_loop2 swap (1.970±0.153 s−1), THAS1 Y56S (0.118±0.005 s−1) and THAS1 E59A (0.061±0.005 s−1).Since the major sequence and structural difference between HYS and THAS1 is the extended loop over the HYS active site, loop 2 , we swaphemistry . Mutatiohemistry . Mutatiohemistry , perhapshemistry . HoweverC. roseus cells. Microscopy of transiently transformed cells revealed that THAS2–YFP , that is not expected to interact with SGD , we noted the formation of a dual fluorescent signal for THAS2, both in the cytosol and as punctates in the nucleus that may correspond to the superposition of the signal observed for THAS2 self-interactions and THAS2–SGD interaction  as confiin planta the genes encoding active HYSs were silenced. For many medicinal plants, including C. roseus, virus-induced gene silencing (VIGS) is the only established method to silence genes in the whole plant. In C. roseus, the effect of VIGS is temporally and spatially limited to the first two leaves that emerge immediately after infectionTHAS1, THAS2, THAS3, THAS4 and HYS) was subjected to VIGS in C. roseus seedlings and the effect on alkaloid production was monitored by MS. Since HYS and THAS4 were too similar to silence separately, one common gene fragment was used for silencing both genes simultaneously. Successful silencing of the genes was confirmed by quantitative reverse transcription–PCR (qRT–PCR) (THAS2 and THAS3 (12%), all of the target genes were silenced selectively, as measured by qRT–PCR  . Aside f qRT–PCR .in planta are difficult to accurately detect. Instead, the effect of silencing must be established by quantitatively measuring decreases in heteroyohimbine levels. Previously for THAS1, we measured the combined peak for heteroyohimbines, since the diastereomers were difficult to resolve on a reverse phase LC column under the reported conditionsC. roseus leaves, THAS2 and THAS3 were silenced. However, for HYS, there was a statistically significant decrease (t-test 0.0275) in ajmalicine, and no change in tetrahydroalstonine levels. Surprisingly, a statistically significant decrease in ajmalicine, as well as tetrahydroalstonine  was also noted for THAS1 , changes in the level of this compound or THAS1 . While tin planta  and the MDR sequences with the highest identity to THAS1 at the active site and which showed non-negligible expression levels in young and mature leaves were selected as candidates for cloning and expression. The protein sequence of SAD was blasted against the same database and MDRs were also selected based on their active site similarity to that of SAD. The genes coding the candidate MDRs were amplified from C. roseus leaf cDNA and cloned into the E. coli expression vector pOPINF using the In-Fusion cloning kit (Clontech Takara)The nucleotide and the protein sequences of THAS1 were subjected to a BLAST search against the E. coli Stellar strain cells (Clontech Takara). HYS point mutants were obtained as gene fragments  with the H127 or F128 codons mutated  and the pOPINF overhangs included at the 3′ and 5′ extremities. The THAS1 and HYS double loop mutants were generated by first making their loop 1 mutant genes and then inserting the second loop 2 swap following the same procedure described above. Mutant constructs were sequenced to verify the mutant gene sequence and correct insertion.THAS1 mutants were generated by overlap extension PCR. Briefly, the codon to be mutated was selected and two primers, one reverse and one forward , were deE. coli cells (Genlantis) grown in 2 × YT medium. Protein production was induced by addition of 0.2 mM IPTG and the cultures were shaken at 18 °C for 16 h. Cells were collected by centrifugation, lysed by sonication in Buffer A  supplemented with EDTA-free protease inhibitor (Roche Diagnostics) and 0.2 mg ml−1 lysozyme. Soluble proteins were purified on Ni-NTA agarose (Qiagen) and eluted with Buffer B . Eluates were analysed by SDS–PAGE to verify the purity and the molecular weight of the purified proteins. All proteins were dialysed in Buffer C  and concentrated. Protein concentration was measured with Bradford reagent (Sigma-Aldrich) according to the manufacturer's instructions. Purified proteins were divided in 20 μl aliquots, fast-frozen in liquid nitrogen and stored at −20 °C.All candidate enzymes and mutants were expressed in SoluBL21 (DE3) 2O+0.1% formic acid) and centrifuged for 10 min at 4,000g before UPLC-MS injection (1 μl). The activity of MDR enzymes and mutants was measured by UPLC-MS. Enzymes exhibited a loss of activity after one freeze thaw cycle.Candidate MDR enzymes and the selected mutants were screened for activity against deglycosylated strictosidine. The substrate was generated by deglycosylating strictosidine (300 μM) by the addition of purified SGD in the presence of 50 mM phosphate buffer (pH 6.5) at room temperature for 10 min. The reactions were started by the addition of MDR enzyme (1 μM) and NADPH (5 mM). Caffeine (50 μM) was used as internal standard. All reactions were performed in triplicate. Aliquots of the reaction mixtures (10 μl) were sampled 1 and 30 min after addition of MDR enzyme. The reactions were stopped by the addition of 10 μl of 100% MeOH. Samples were diluted 1:5 in mobile phase , as well as for the mutants THAS1 Y56S, E59A and HYS loop 2 swap. Reactions were monitored at 340 nm at room temperature using a  spectrophotometer. Strictosidine (50 μM) was incubated with purified SGD in 50 mM phosphate buffer (pH 7.0) for ∼30 min at 30 °C. NADPH (100 μM) was added, mixed by pipetting and the absorbance at 340 nm was monitored until stabilized (minimum of 2 min). A predetermined amount of enzyme (10–400 nM) was then added, mixed and the reaction was monitored for a minimum of 5 min at 340 nm. The resulting slope was calculated during the linear reaction range, usually 0–60 s after addition of the enzyme. The reactions were replicated five times and the initial velocity was calculated for each replicate after accounting for the background. The concentration of substrate (50 μM strictosidine) was determined to be saturating for all wild-type enzymes under these conditions.g for 20 min. Two-dimensional automated purification was performed on an AKTAxpress purifier . The IMAC step was performed on HisTrap HP 5 ml columns  equilibrated with Buffer A. Proteins were step-eluted with Buffer B and directly injected on a gel filtration column equilibrated with Buffer D . Fractions were collected and analysed by SDS–PAGE and those containing pure protein were pooled and concentrated in a 10 kDa membrane filter—Millipore filter (Merck Millipore).Proteins for crystallization were purified from 2 l cultures in 2 × YT medium. Protein expression was induced by addition of IPTG and the cultures were grown for 16 h at 18 °C. Cells were collected by centrifugation and lysed by sonication in 50 ml Buffer A supplemented with EDTA-free protease inhibitor and 10 mg of Lysozyme. Lysates were clarified by centrifugation at 17,000tris(2-carboxyethyl)phosphine (TCEP) before crystallization and storage.Purification of HYS required the addition of 1 mM DTT to all purification buffers and dialysis in Buffer D containing 0.5 mM −1 while NADP+ (Sigma-Aldrich) was added to a final concentration of 1 mM for co-crystallization studies. Solutions were dispensed either by an OryxNano or an Oryx8 robot (Douglas Instruments).Crystallization screens were conducted by sitting-drop vapour diffusion in MRC2 96-well crystallization plates (Swissci) with a mixture of 0.3 μl well solution from the PEGs (Qiagen), PACT (Qiagen) and JCSG (Molecular Dimensions) suites and 0.3 μl protein solution. Protein concentrations were adjusted to 7–10 mg ml6-tag cleaved THAS1 (3C protease) in a solution containing 0.1 M MES, pH 6.5, 15% w/v PEG 2000. THAS1-NADP+ crystals were obtained from a solution containing 0.2 M potassium/sodium tartrate with 20% w/v PEG 3350. THAS2 crystals (with and without NADP+) were obtained from a condition containing 0.2 M lithium chloride and 20% w/v PEG 3350. HYS crystals were obtained after removal of the His6-tag (using 3C protease) in 0.1 M MMT buffer, pH 5 and 15% w/v PEG 3350. All crystals were cryoprotected by soaking in crystallization solution containing 25% v/v ethylene glycol before flash-cooling in liquid nitrogen.THAS1 apo crystals were obtained from His+ data set was automatically processed at the beamline by fast_dpK X-ray absorption edge , the anomalous signal was sufficient for fast_ep to locate four zinc sites and calculate a very clear experimentally phased electron density map  before running PHASER. For solving the THAS2 apo structure, a THAS2 NADP+ monomer was used as the template. In contrast to THAS1-NADP+, these four structures were refined in REFMAC5 with isotropic thermal parameters and TLS group definitions obtained from the TLS-MD serverRpim: 5FI3, 0.020 (0.517); 5FI5, 0.038 (0.600); 5H81, 0.041 (0.349); 5H82, 0.033 (0.439); 5H83, 0.068 (0.664) and CC½: 2FI3, 0.999 (0.510); 2FI5, 0.999 (0.523); 5H81, 0.998 (0.725); 5H82, 0.999 (0.639); 5H83, 0.996 (0.510)  were also noted. Ramachandran statistics ) are 5FI3, 96.8/3.2/0.0; 5FI5, 96.0/4.0/0.0; 5H81, 96.2/3.8/0.0; 5H82, 96.1/3.9/0.0; 5H83, 96.6/3.1/0.3. All structural figures were prepared using CCP4mgX-ray data sets were recorded on one of three beamlines at the Diamond Light Source   at wavelengths of 0.9000–0.976 Å  using either a Pilatus 6M or 2M detector (Dectris) with the crystals maintained at 100 K by a Cryojet cryocooler (Oxford Instruments). Diffraction data were integrated using XDSsity map . This wasity map  against sity map , and conhttps://www.phenix-online.org) from a starting temperature of 5,000 K after applying small random shifts to the model (‘shake' term set to 0.3). The resultant mFobs–dFcalc difference electron density maps (contoured at ∼3.0σ) are displayed superposed on the final coordinates, where the corresponding omitted atoms are shown in stick representation. In each case, structurally equivalent residues were omitted, with the exception of HYS where His127 from loop 2 was also omitted.Simulated annealing omit maps were calculated for the active site regions of all five structures presented in this study. For all structures, selected residues bordering the active site (and the cofactors in the case of the two holoenzyme structures) were deleted from the coordinates of the final models. The resultant PDB files were used as inputs to simulated annealing refinement with PHENIX  equipped with an Acquity BEH C18 1.7 μm 2.1 × 50 mm column connected to Xevo TQS (Waters). MS detection was performed in positive ESI. Capillary voltage was 3.0 kV; the source was kept at 150 °C; desolvation temperature was 500 °C; cone gas flow, 50 l h−1; and desolvation gas flow, 800 l h−1. Unit resolution was applied to each quadrupole. Multiple reaction monitoring signals were used for detection and quantification of caffeine  and heteroyohimbine alkaloids .Enzyme assays and plant tissue samples from VIGS experiments on −1 using a binary solvent system in which solvent A1 was 0.1% formic acid in water and solvent B1 was acetonitrile. The gradient profile was: 0 min, 5% B1; from 0 to 3.5 min, linear gradient to 35% B1; from 3.5 to 3.75 min, linear gradient to 100% B1; from 3.75 to 4 min, wash at 100% B1; back to the initial conditions of 5% B1 and equilibration for 1 min before the next injection. Column temperature was held at 30 °C. The injection volume for both the solutions of standard compounds and the samples was 1 μl. Samples were kept at 10 °C during the analysis.For rapid dereplication of active enzymes and mutants, a linear gradient method (Method 1) was used at a flow rate of 0.6 ml minet al.4OH and solvent B2 was 0.1% NH4OH in acetonitrile. A linear gradient from 0 to 65% B2 in 17.5 min was applied for separation of the compounds followed by an increase to 100% B2 at 18 min, a 2-min wash step and a re-equilibration at 0% B2 for 3 min before the next injection. The column was kept at 60 °C throughout the analysis and the flow rate was 0.6 ml min−1.For separation of the different heteroyohimbines, a different chromatographic method was applied that was adapted from the work of Sun J. R-NADPD was regenerated in solution by Thermoanaerobacter brockii alcohol dehydrogenase  using 400 μM NADP+ and 1% v/v [2H6]-isopropanol (CIL). The NADPD regeneration was monitored by ultraviolet spectroscopy at 340 nm. Strictosidine (19.9 mg) was incubated with 1.27 nM SGD in 94 ml of 50 mM phosphate buffer (pH 6.5). THAS1 enzyme was added to the reaction  and the mixture was incubated at 35 °C. The reaction was monitored for completeness by UPLC-MS and after 5 h no strictosidine or deglycosylated strictosidine was observed. The reaction was stopped by addition of 100 ml of methanol and reaction mixture was concentrated to dryness. The dried reaction mixture was resuspended in 15 ml H2O and extracted with 3 × 15 ml of ethyl acetate and the EtOAc fraction was dried. [21α-2H1]-tetrahydroalstonine was isolated by preparative TLC , as previously described2H1]-tetrahydroalstonine was excised from the plate and THA was extracted with EtOAc multiple times . The EtOAc fraction was filtered and dried under high-vacuum overnight. The [21α-2H1]-tetrahydroalstonine was dissolved in 600 μl of CDCl3 and 1H-NMR was measured.Deuterated Pro-+ with 50 U of T. brockii ADH and 1% v/v [2H6]-isopropanol in a total volume of 148 ml of 50 mM HEPES buffer (pH 7.5). HYS was added  and the reaction was incubated at 37 °C with shaking and monitored for completeness by UPLC-MS. After 6 h, the reaction was complete and was stopped by addition of 150 ml of methanol. [21α-2H1]-tetrahydroalstonine, [21α-2H1]-ajmalicine and [21α-2H1]-mayumbine were isolated by preparative TLC and 1H-NMR spectra measured as described above.Strictosidine (39.3 mg) was incubated with 1 nM of SGD and 500 μM NADPC. roseus tryptophan decarboxylase (TDC) was cloned into pOPINF vector, expressed in E. coli and purified as described above for the MDRs. [alpha-15N]-tryptophan  was incubated with 500 nM of TDC, 400 μM pyridoxal-5′-phosphate in 100 ml 50 mM phosphate buffer (pH 7.5) at 35 °C. The reaction was monitored by MS, continued through completion after 4 h and terminated by addition of 50 ml MeOH. [alpha-15N]-tryptamine (34 mg) was isolated by preparative HPLC. The isolated [alpha-15N]-tryptamine was incubated with 3 mM secologanin and 200 nM strictosidine synthase in 100 ml 50 mM phosphate buffer (pH 7.0) at 30 °C overnight. The reaction was terminated by addition of 50 ml MeOH. [4-15 N]-strictosidine (62 mg) was isolated by preparative HPLC. [4-15N]-strictosidine was then assayed with SGD and the product was extracted the ethyl acetate, dissolved in MeOH-d4 and characterized by 1H,15N-HMBC as described above.1H equipped with a BBFO plus 5-mm probe. The number of scans depended on the concentration of the sample. The 1H,15N-HMBC experiment was acquired with a spectral width 6,009 Hz in the F2 (1H) dimension and 30,410 Hz in the F1 (15N), with an acquisition time of 0.09 s and 360 scans per increment. The long range delay was optimized after a series of experiments with [4-15N]-strictosidine using a range of different mixing times and finally was adjusted for a coupling of 5 Hz. The relaxation delay was 2.5 s, the data collection matrix was 1024 × 64, the t1 dimension was zero filled to 1k real data points and a π/2 square sine bell window was applied in both dimensions. The 1H-NMR spectra were compared with those of standards and literature data.High-resolution electrospray ionization MS spectra were measured with a Shimadzu IT-TOF mass spectrometer. NMR spectra were acquired using a Bruker Advance NMR instrument operating at 400 MHz for [21α-2H1]-tetrahydroalstonine. TLC : Rf=0.58; HR-MS (IT-TOF): found for [M+H]+: [C21H24DN2O3]+=354.1921: calcd 354.19225; 1H-NMR : δ 7.80 , 7.56 , 7.45 , 7.28 , 7.12 , 7.07 , 4.50 , 3.74 , 3.36 , 3.08 , 2.95 , 2.89 , 2.77 , 2.69 , 2.56 , 2.49 . 1.70 , 1.53 , 1.40 .[21α-2H1]-ajmalicine. TLC : Rf=0.41; HR-MS (IT-TOF): found for [M+H]+: [C21H24DN2O3]+=354.1921: calcd 354.19225; 1H-NMR : δ 7.94 , 7.53 , 7.46 , 7.30 , 7.13 , 7.08 , 4.43 , 3.74 , 3.41 , 3.22 , 3.10 , 3.00 , 2.95 , 2.75 , 2.68 , 2.42 , 2.14 , 1.32 , 1.19 .[21α-2H1]-mayumbine. TLC : Rf=0.45; HR-MS (IT-TOF): found for [M+H]+: [C21H24DN2O3]+=354.1921: calcd 354.19225; 1H-NMR : δ 7.96 , 7.56 , 7.46 , 7.30 , 7.13 , 7.09 , 3.88 , 3.73 , 3.40 , 3.16 , 3.14 , 3.10 , 3.03 , 2.72 , 2.40 , 1.76 , 1.37 , 1.30 , 1.21 .[4-15N]-strictosidine. HR-MS (IT-TOF): found for [M+H]+: [C27H35N15NO9]+=532.2304: calcd 532.23074; 1H-NMR : δ 10.5 , 7.77 , 7.44 , 7.30 , 7.11 , 7.02 , 5.84 , 5.83 , 5.34 , 5.26 , 4.79 , 3.96 , 3.78 , 3.64 , 3.42–3.30 , 3.23 , 3.22 , 3.10-3.00 , 2.97 , 2.72 , 2.27 , 2.18 ; 15N-NMR : δ 44.8.SpeI restriction site at both cDNA extremities. PCR products were sequenced and cloned at the 5′ end of the YFP-coding sequence to generate the THAS2–YFP, HYS–YFP fusion proteins or at the 3′ end to express the YFP–THAS2 and YFP–HYS fusions.Subcellular localization of THAS2 and HYS were studied by creating fluorescent fusion proteins using the pSCA-cassette YFPi plasmidSpeI into the pSPYCE (M) vectorC fragment , and into the pSPYNE(R)173-SGD plasmidN fragment (YFPN–SGD). Plasmids encoding THAS1–YFPN, THAS1–YFPC, YFPC–THAS1 and plasmids expressing 16OMT–YFPN and 16OMT–YFPC were used as controls and were constructed previously24The interaction of THAS2 and HYS with SGD were characterized by BiFC assays using the previously amplified THAS2 and HYS PCR products cloned via R)173 and pSCA-SPYCE (MR) plasmids34N, HYS–YFPC and YFPC–THAS2, YFPC–HYS, respectively.THAS2 and HYS self-interactions were analysed via additional cloning of the THAS2 and HYS PCR products into the pSCA-SPYNE173, pSPYNE and the CFPN fragment (CFPN-SGD).The capacities of THAS2 and HYS to interact with SGD were also characterized by double BiFC and mBIFC. The previously amplified THAS2 and HYS PCR products were fused to the coding sequences of the amino-terminal or carboxy-terminal of the split YFP fragments into the pSCA-SPYNE173, pSCA-SPYCE (M) and pSCA-SPYCE (MR) plasmids34C. roseus cells by particle bombardment and fluorescence imaging were performed following the procedures previously describedC. roseus-plated cells were bombarded with DNA-coated gold particles (1 μm) and 1,100 psi rupture disc at a stopping-screen-to-target distance of 6 cm, using the Bio-Rad PDS1000/He system. Cells were cultivated for 16–38 h before being harvested and observed. The subcellular localization was determined using an Olympus BX-51 epifluorescence microscope equipped with an Olympus DP-71 digital camera and a combination of YFP and CFP filters. The pattern of localization presented in this work is representative of circa 50 observed cells. The nuclear localizations of the different fusion proteins were confirmed by co-transformation experiments using a nuclear-CFP markerTransient transformation of C. roseus seedlings essentially as described beforeg, an aliquot of the supernatant (20 μl) was diluted to 200 μl with methanol, filtered through 0.2-μm PTFE filters and analysed on Waters Xevo TQ-MS. The chromatographic separation and MS measurements were carried out as described above (method 2). To more comprehensively assess the global effect of silencing the HYS genes by VIGS on C. roseus metabolism, an untargeted metabolomics analysis by LC-MS was performed as previously reported.The THAS1, THAS2, THAS3 and THAS4-HYS silencing fragments were amplified with primers  and the Gene silencing was confirmed by qRT–PCR. qRT–PCR was also used to check the expression of the other HYS genes to ensure that no cross-silencing occurred. RNA extraction was performed using the RNeasy Plant Mini Kit (Qiagen). RNA (1 μg) was used to synthesize cDNA in 20-μl reactions using the iScript cDNA Synthesis Kit (Bio-Rad). The cDNA served as template for quantitative PCR performed using the CFX96 Real Time PCR Detection System (Bio-Rad) using the SSO Advanced SYBR Green Supermix (Bio-Rad). Each reaction was performed in a total reaction volume of 20 μl containing an equal amount of cDNA, 0.25 mM forward and reverse primers and 1 × Sso Advanced SYBR Green Supermix (Bio-Rad). The reaction was initiated by a denaturation step at 95 °C for 10 min followed by 41 cycles at 95 °C for 15 s and 60 °C for 1 min. Melting curves were used to determine the specificity of the amplifications. Relative quantification of gene expression was calculated according to the delta–delta cycle threshold method using the 40 S ribosomal protein S9 (RPS9). All primer pair  efficienStrictosidine was deglycosylated using purified SGD for 25 min at room temperature using assay conditions as described above. Strictosidine aglycone was then incubated at a final concentration of 300 μM at pH 5, 6, 7 and 8 in a buffer mix to avoid buffer ingredient effect on activity . Caffeine (50 μM) was used as an internal standard.4 , was added to deglycosylated strictosidine as a control reaction. All reactions were carried out in triplicate. An end-point sample (10 μl) was taken for each assay and prepared for UPLC-MS by addition of 10 μl of 100% MeOH to stop the reaction, and then diluted 1:5 with H2O, and centrifuged for 10 min at 4,000 r.p.m. UPLC-MS and data collection were performed as described above for heteroyohimbine separation and quantification.At time 0 the enzyme, either THAS1 or HYS , premixed with NADPH (500 μM) was added to the substrate solution. In parallel, a chemical reducing agent, NaBHFar ultraviolet CD spectra of the wild-type enzymes THAS1 and HYS, as well as the loop mutants of THAS1 and HYS were recorded on a Chirascan Plus spectropolarimeter (Applied Photophysics) at 20 °C in 10 mM potassium phosphate buffer pH 7.0. Samples were analysed from 180 to 260 nm using a 0.5-nm step at a speed of 1 s per step. Four replicate measurements were performed on each sample and baseline correction was applied to all data. Spectra are presented as the CD absorption coefficient calculated on a mean residue ellipticity basis.−1 from 20 to 90 °C. Data collection was done from 260 to 201 nm using a 1-nm step and 0.75 s time per point. Data were analysed using the Global 3 software. HYS melting point was measured as 61.0±0.1 °C; enthalpy 351.5±3.6 KJ mol−1. HYS loop 2 swap melting point was measured at 62.0±0.1 °C; enthalpy 535.8±4.5 KJ mol−1.Melting curves of HYS and the HYS loop 2 swap mutant were also acquired by CD. The samples were subjected to temperature ramping at the rate of 1 °C minhttp://www.geneious.com)62http://espript.ibcp.fr)Protein sequence alignment was generated using ClustalW algorithm with Geneious v.8 , as selected by the software, is used in the structures illustrated here (Cathenamine was docked into the THAS1-NADP4.2 ref. . The lig4.2 ref. . The posted here .http://www.pdb.org/), with accession codes 5FI3, 5FI5, 5H81, 5H82 and 5H83. Accession numbers: THAS1 (AKF02528.1); THAS2 (KU865323); THAS3 (KU865322); THAS4 (KU865324); HYS (KU865325); Cro_017994 (KU865326); Cro_011702 (KU865327); Cro_030442 (KU865328); Cro_006840 (KU865329); Cro_022770 (KU865330); Cro_033537 (KU865331); Cro_027234 (AHK60846); Cro_033062 (KU865332); Tabersonine-3-reductase (AKM12281). Data supporting the findings of this study are available within the article and its Supplementary Information files and from the corresponding author upon reasonable request.The atomic coordinates and structure factors of the five X-ray structures described in this manuscript have been deposited in the Protein Data Bank .Supplementary Figures 1-18, Supplementary Tables 1-8 and Supplementary References.


We report a new strategy for generating a continuum of response profiles from a single luminescence-based sensor element by using phase-resolved detection. This strategy yields reliable responses that depend in a predictable manner on changes in the luminescent reporter lifetime in the presence of the target analyte, the excitation modulation frequency, and the detector (lock-in amplifier) phase angle. In the traditional steady-state mode, the sensor that we evaluate exhibits a linear, positive going response to changes in the target analyte concentration. Under phase-resolved conditions the analyte-dependent response profiles: (i) can become highly non-linear; (ii) yield negative going responses; (iii) can be biphasic; and (iv) can exhibit super sensitivity . Inspection of i.e., creating diversity) from a single sensor element by adjusting θD and f.2.2.1.3]2+) was purchased from GFS Chemicals, Inc.  and purified as described in the literature [n-octyltriethoxysilane (C8-TEOS) were purchased from Gelest, Inc. . HCl was obtained from Fisher Scientific Co. . EtOH was a product of Quantum Chemical Corp. . Deionized water was prepared to a specific resistivity of at least 18 MΩ-cm by using a Barnstead NANOpure® II system.Tris ruthenium(II) chloride pentahydrate ([Ru(dpp)terature . Tetraet2.2.sol solutions were prepared as described elsewhere [max = 468 nm, Stanley Electric Co., Inc., . The LED surface was cleaned first by rinsing with 1 M NaOH then with copious amounts of deionized water and EtOH, and allowed to dry under ambient conditions. Films were formed by depositing 20 μL of sol solution on to the LED surface. Xerogel-coated LEDs were stored in the dark under ambient conditions for at least seven days before evaluation. Previous research [I0/I vs. O2 plots and the responses are stable for several years.The lsewhere . Xerogel2.3.2. The emission was monitored through a 570 nm long pass optical filter  and detected by a 1 MHz bandwidth silicon photodiode . The photodiode output signal was directed to the lock-in amplifier input. The lock-in amplifier served to record the phase sensitive luminescence intensity (PSLI) at a given detector phase angle setting (θD).f) to a given value (20 or 50 kHz) and increment θD in 22.5° steps from 0° to 180°. We then systematically adjust the environment surrounding the sensor from pure N2 to pure O2 by using a pair of precision mass flow controllers . Equilibrium is evident when the luminescence intensity remains constant to within ±2%. There was no detectable sensor response hysteresis or photo-bleaching. The O2 concentrations were accurate to ±0.8% between 10% and 100% full scale and to ±0.08% below 10% full scale.In a typical experiment, we adjust the excitation modulation frequency  concentration and θD. In the simulations we assumed: τ0 = 5.0 μs and KSV = 0.1 O2%−1. D. D = 90° in D = 67.5° in D. D = 90° in D = 67.5° in f and θD. Thus, one can tune the sensor response and sensitivity simply by adjusting f or θD. In all previous sensor research, there was no provision for achieving a diverse response from a single sensor element  is ultra-sensitive. For example, in our previous work, the I0/I for 70% O2 is ∼8 for this sensor element [D of 90°. Similarly, at 50 kHz with a θD of 67.5° we see a PSLI0/PSLI at 100% O2 of ∼50 whereas the I0/I at 100% O2 is ∼10 for this same sensor [Inspection of these data sets shows several interesting results. First, the simulations and experiments agree very well. Second, the response profiles depend on ent cf., . Third, .1 O2%−1 . Fourth, element . In Figue sensor .4.We report on a simple and new approach for creating diversified sensor responses from a single sensor element. The approach is not only limited to quenchometric-based sensing, but also will work under any situation where there is a change in a luminophore excited state lifetime caused by the target analyte –25. Phas


The synthesized organic chromophore exhibited a broad absorption peak near green region and strong emission peak due to the presence of strong electron-withdrawing nature of two nitrile (–CN) groups of fumaronitrile acceptor. The highest occupied molecular orbital (HOMO) energy level of –5.82 eV and the lowest unoccupied molecular orbital (LUMO) energy level of –3.54 eV were estimated for RCNR due to the strong electron-accepting tendency of –CN groups. The fabricated SMOSC devices with RCNR:PC The development of organic conjugated donor–acceptor–donor (D–A–D) systems composed of oligothiophenes (donor) and electron-deficient molecule (acceptor) provides an efficient approach since these molecules could increase an absorption band through intramolecular charge transfer (ICT) for better match-up of the solar spectrum and thus, attain the high PCE in organic solar cells devices1718In general, the presence of –CN group in organic polymers lowers HOMO and LUMO values compared to without –CN-groups analogues2021222324252 atmosphere. The TGA plot (d) of the RCNR is found as ~368 °C, indicating a relatively high thermal-stability of the organic chromophore which is expedient for the solution-processed device fabrication and the operation of organic solar cellsm) at ~69 °C, ~161 °C, and ~172 °C, with no signs of a glass-transition temperature (Tg), while an isotropic transition phase is observed after ~270 °C3436The thermal stability of the synthesized organic chromophore, RCNR has been analysed by the thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) under NTGA plot  reveals −5 M) and thin film state. In chloroform solution, the two distinct peaks are observed. The spectra shows a relatively small absorption peak at λmax ≈ 368 nm and another broad absorption peak at λmax ≈ 465 nm. The molar absorption coefficient (ε) in solution is calculated as ~1.58 × 104 M−1 cm−1 which indicates a strong intramolecular charge transfer (ICT) interaction behavior between thiophene donor and fumaronitrile-acceptor3841gopt) of ~2.03 eV is calculated by the absorption edge (λedge) from solid thin film absorption by the formula:UV-Vis absorption spectra  of RCNR 60BM acceptor, suggesting the electron transfer from donor to acceptor and the fast charge-transfer which is enough to compete with the radiative recombination of the excitons44The photoluminescensce spectra  of the s3CN solution of tetrabutyl ammonium hexa flouro phosphate [nBu4N]+[PF6]− at a potential scan rate of 100 mV/s. The oxidation and reduction peaks are situated at the onset value of Eox = +1.42 ± 0.02 eV and Ered = –0.86 ± 0.02 eV. Hence, the RCNR solid thin film exhibits HOMO and LUMO of –5.82 eV and –3.54 eV, respectively. The observed electrochemical band gap is found to be Egel = 2.28 eV. The difference of HOMO and LUMO energy level is a crucial factor for determining the energy band gap which indicates the electrons delocalization in the solid thin films46The redox properties of the organic chromophore are measured by cyclic voltammetry (CV) studies of RCNR thin film  in 0.1 M61-butyric acid methyl ester (PC60BM) as an electron-acceptor with a standard device structure of ITO/PEDOT:PSS (~80 nm)/RCNR:PC60BM blend (~60 nm)/Ag (~100 nm). The blended active layers of the solar cell devices are developed by spin-casting the various  mixtures of the RCNR with PC60BM. The photovoltaic properties . The PCE of ~2.69% is achieved by the SMOSC devices fabricated with RCNR:PC60BM  active layer ratio, whereas the other fabricated SMOSC devices exhibit inferior PCEs of ~1.50% for RCNR:PC60BM , ~2.0% for RCNR:PC60BM  and ~2.23% for RCNR:PC60BM  active layer ratios. The SMOSC fabricated with RCNR:PC60BM  active layer presents the JSC of ~9.68 mA/cm2, and high VOC of ~0.792 V. Herein, the presence of –CN groups connecting with vinyl double bond enhances the conjugation length of chromophore and hence, better electron-delocalization which might affect the open-circuit voltage and short-circuit density of the solar cell devicesOC value at low concentration ratios  of RCNR in the blended active layers.Solution-processed BHJ small molecule organic solar cells are fabricated using RCNR as an electron-donor and -phenyl Coperties  of the foperties  under thDOT:PSS ~0 nm/RCNR60BM  thin films, as shown in 60BM  blended active layer (rms = 2.06 nm) in nanoscale phase separation which contributes to good miscibility of donor-acceptor, high exciton-dissociation rate and better charge transport. On the other hand, other blended active layers of RCNR:PC60BM  record high Rrms values of 9.20 nm, 2.63 nm, 3.29 nm, respectively. These results show that the RCNR:PC60BM  active layer is the optimized one for homogeneous miscibility between donor and acceptor yielding a smooth thin film morphology and a large donor-acceptor (D−A) interface area with nanoscale phase separation60BM  film depicts the lowest Rrms of ~2.06 nm as compared to other blended RCNR:PC60BM  active layers, suggesting the homogenous nature of RCNR and PC60BM molecules in the blended layer which provides enough surface area for exciton-dissociation60BM  device shows a minimum series-resistance and hence, a maximum FF of ~0.35. Furthermore, the lower values of FF are related to increase in the series-resistance of RCNR:PC60BM/ITO in SMOSCs, resulting in a higher recombination rate over the surface of RCNR:PC60BM blended active layers60BM, a bicontinuous network structure might form which creates the percolation channels for the efficient charge carrier collection within the active layer of BHJ solar cellsOC value might be due to the presence of two –CN groups which induce the better film morphology and strong intermolecular charge-transfer (ICT) between RCNR and PC60BM960BM which improves the film-morphology of the blended active layers and ultimately increases the photocurrent-density for the better performance of solar cell devicesThe atomic force microscopy (AFM) analysis is used to investigate the morphological behavior of the blended active layer RCNR:PCve layer  clearly 6) in acetonitrile as the supporting electrolyte. The RCNR was dissolved in chloroform solvent and thin film was deposited on the glassy carbon working electrode by drop casting and dried at 60 °C for 4 h under nitrogen. NMR spectra were obtained in CDCl3 solvent (1H at 600 MHz and 13C at 100 MHz) using JEOL FT-NMR spectrophotometer. For NMR analysis, CDCl3 was used as the solvent and chemical shift values  were considered in parts per million (ppm) with tetramethylsilane (TMS) as the internal reference. Thermo-gravimetric analysis (TGA) was carried out with a TA instruments Q-50 thermogravimetry analyzer at a scan rate of 10 °C/min under inert atmosphere. The differential scanning calorimetry (DSC) was characterized by TA instrument DSC-2910 at heating rate of 10 °C/min under nitrogen atmosphere.Unless otherwise noted, the chemicals and reagents were purchased from commercial sources as Sigma–Aldrich, Alfa-aesar and TCI chemical companies and used as received. Thin layer chromatography (TLC) was performed on Merck TLC-plates of aluminum coated with silica gel 60 F254. Flash column chromatography was performed on a column packed with silica gel (300–400 mesh). Fourier transform-infrared (FTIR) spectroscopy was performed by FT/IR-4100 (JASCO) spectrometer. Ultra violet–visible (UV-vis) absorption and photoluminescence spectra (PL) were recorded by V-670 (JASCO) spectrophotometer and FP-6500 (JASCO) fluorometer, respectively. The cyclic voltammetry (CV) measurements were done using WPG 100 Potentiostat/Galvanostat (WonATech) at a scan rate of 50 mV/s with a three-electrode cell consisting of a glassy carbon working electrode, a saturated calomel reference electrode (SCE) and a platinum wire as counter electrode. CV measurement was performed in 0.1 M solution of tetrabutylammonium hexafluorophosphate 4 (2.5 mol %) as catalyst and potassium carbonate, K2CO3 as a base in anhydrous toluene solvent under inert atmosphere. The synthesized red-colored chromophore was then purified by repeated crystallization in the mixed solvent of dichloromethane/methanol  with a decent yield of 86.4%.The reaction intermediates 1  in anhydrous benzene (20 mL) at the room temperature. Then TiCl4  was added slowly to the reaction mixture at 0°C and was stirred for 15 min at 0 °C. After completion of the reaction, cold water was added into the reaction mixture to quench the reaction. The resulting mixture was diluted with CH2Cl2 (50 mL), washed successively with water (200 mL) and saturated aqueous solution of NaHCO3 (100 mL), then dried over MgSO4 followed by an evaporation under vacuum to afford a yellow solid , anticipated as the desired ketone intermediate 2 which was used directly for next step of the reaction.Hexanoyl chloride  was added to a solution of 2,2’-bithiophene 2  in anhydrous toluene (40 mL) and a suspension of LiAlH4  and AlCl3  in anhydrous Et2O (100 mL) were mixed slowly at 0 °C with extreme care. The reaction mixture was then stirred for 1 h at the room temperature, and again cooled at 0 °C, then ethyl acetate (20 mL) and HCl (6 M) solution (50 mL) were added to the reaction mixture. The resulting mixture was extracted with diethyl ether (300 mL), washed with NaCl solution and distilled water (50 mL) thereafter, dried over MgSO4. Afterward, solvent of the organic phase was evaporated by using rotatory evaporator followed by drying in the vacuum. The yellow residue was then purified by flash column chromatography on silica gel (hexane) to produce compound 3  as a colourless oil. 1H NMR : 7.15 , 7.10 , 6.96 , 6.95 , 6.65 , 2.75 , 1.69 , 1.35 , 0.90 ; 13C NMR : 146.4, 145.2, 138.0, 134.8, 127.7, 125.7, 124.3, 123.8, 123.5, 123.0, 32.0, 31.7, 30.2, 29.5, 29.4, 29.2, 22.8, 14.2.Under nitrogen atmosphere, the solution of intermediate 3  in dimethylformamide (30 mL) and the reaction mixture was stirred for 30 min in the absence of light then, diluted with hexane (50 mL), washed with saturated aqueous solution of NH4Cl (50 mL), dried over MgSO4, and evaporated under vacuum. The residue was purified by column chromatography on silica gel (hexane) to give a white solid compound 4 . 1H NMR : 6.95 , 6.90 , 6.85 , 6.68 , 2.78 , 1.66 , 1.35 , 0.90 ; 13C NMR : 145.6, 139.9, 133.5, 130.5, 130.1, 124.8, 123.7, 123.1, 110.2, 32.2, 31.7, 30.3, 29.9, 29.6, 29.4, 22.9, 14.4.N-bromo succinimide  was added to a solution of compound 4  and tetrahydrofuran (20 mL) was added to n-BuLi  at –78 °C. The temperature was increased slowly up to –50 °C within 20 min. 2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane  was added and the temperature was increased slowly to the room temperature. The reaction mixture was then stirred for 3 h at room temperature and 2N HCl (20 mL) was added. The resulting mixture was extracted with diethylether (30 mL), washed with NaCl solution followed by distilled water (500 mL), dried over MgSO4, and evaporated under vacuum. The obtained residue was recrystallized from hexane (10 mL) to yield a white solid compound, 5 . 1H NMR : 7.55 , 7.20 , 7.05 , 6.70 , 2.81 , 1.72 , 1.38 , 0.91 ; 13C NMR : 146.1, 144.8, 137.9, 134.7, 124.9, 124.5, 124.1, 84.1, 31.9, 31.5, 30.2, 29.6, 29.4, 29.3, 24.8, 22.6, 14.3.Under nitrogen atmosphere, a solution of compound 6  and iodine  was purged with N2 and subsequently anhydrous diethyl ether (100 ml) was injected via syringe. A solution was cooled to –78 °C. Sodium methoxide  and methanol (40 ml) was added slowly over a period of 30 min and then stirred for 40 min. Then the reaction solution was put to ice-water bath at 0 °C with stirring for further 4 h. Hydrochloric acid (3–6%) was added dropwise to the reaction mixture and the solution was filtered to isolate the precipitate, which was then rinsed with cold methanol-water solution. Filtrate was concentrated further and a second crop of target product was obtained as a pale yellow solid, 7 . FT-IR : 3096, 2220, 1585, 1488, 1396, 1245, 1074, 1007, 845, 816, 710, 665, 627, 573, 514. 1H NMR :7.67-7.72 .A mixture of 4-bromophenylacetonitrile, 5  and monomer 7  with triphenylphosphine  were mixed and then subjected to three cycles of evacuation and nitrogen purging in anhydrous toluene (~10 mL) solvent. Aqueous solution of potassium carbonate  was added by syringe to the reaction mixture and was stirred at 110 °C for 12 h. The reaction mixture was cooled down to the room temperature followed by the addition of water. Subsequently, an organic phase was extracted with dichloromethane (~20 mL) and the reaction mixture was washed with brine and distilled water and dried over magnesium sulfate. The solution was filtered and evaporated in vacuum to achieve a red colored residue, which was then recrystallized several times in dichloromethane and methanol (2:1 v/v) mixture to get organic chromophore as a dark red solid . FT-IR : 3067, 2955, 2926, 2853, 2219, 1631, 1581, 1488, 1396, 1245, 1084, 1007, 845, 816, 710, 665, 627. 1H NMR : 7.89–7.87 , 7.75-7.71 , 7.55–7.52 , 7.36-7.34  7.11-7.02 , 6.72-6.71 , 2.84-2.78 , 1.70-1.68 , 1.39-1.31 , 0.92-0.88 . 13C NMR : 145.2, 139.2, 138.2, 136.3, 133.2, 129.1, 128.6, 128.4, 124.8, 124.6, 124.1, 122.9, 116.2, 115.9, 30.6, 30.4, 29.2, 29.0, 27.8, 27.5, 21.5, 21.3, 12.9; MS: m/z 726 (M+).In a 50 mL round bottom flask, monomer 60BM layer (thickness ~60 nm) with w/w blending ratio of 1:1, 1:2, 1:3 or 1:4 in o-dichlorobenzene solution (10 mg/ml) was again spin-coated on PEDOT:PSS film-coated ITO at a scan rate of ~700 rpm for 40 s. The fabricated active layer was heated at 80 °C for 10 min to evaporate the residual solvent. Finally, the silver cathode (thickness ~100 nm) was deposited through a shadow mask by thermal evaporation under a vacuum of about 3 × 10–6 Torr. The active area of device was measured as ~1.5 cm2. The photovoltaic properties of the cells were measured under simulated AM 1.5 radiation at 100 mW/cm2 using 1000 W metal halide lamp (Phillips) which was served as a simulated sun light source and its light intensity (or radiant power) was adjusted with a Si photo detector fitted with a KG-5 filter (Schott) as a reference, calibrated at NREL (USA). The power conversion efficiency (η) is calculated by the following equation:For the fabrication of SMOSCs, the indium tin oxide (ITO) glass substrate was first cleaned with detergent, ultrasonicated in water, acetone and isopropyl alcohol and subsequently dried overnight in an oven. PEDOT:PSS thin film (thickness ~80 nm) was coated on ITO substrates by spin-coating the solution with a speed of ~4000 rpm for 40 s and thereafter, annealed at 130 °C for 10 min in a vacuum oven. The active RCNR:PCSC is the short-circuit photocurrent density, VOC is the open-circuit voltage, FF is the fill factor, and Pin is the incident radiation power.where J60BM  achieves a reasonable PCE of ~2.69% with JSC of ~9.68 mA/cm2 and VOC of ~0.79 V. The variation in the concentration of PC60BM acceptor in blended active layers has considerably affected the thin film morphology and hence, the performance of the fabricated solar devices.A novel, symmetric D-A-D type fumaronitrile-acceptor based organic π-conjugated chromophore (RCNR) is synthesized and applied as an electron-donor material for the solution-processed fabrication of SMOSCs. The synthesized organic chromophore presents a broad absorption peak near green region and strong emission peak due to the presence of two strong electron-withdrawing −CN groups. The cyclic voltammetry study of RCNR shows relatively deep HOMO of −5.82 eV and LUMO of −3.54 eV, which suggests a strong electron-accepting tendency of –CN groups. The fabricated SMOSC device of active layer RCNR:PCHow to cite this article: Nazim, M. et al. Effective D-A-D type chromophore of fumaronitrile-core and terminal alkylated bithiophene for solution-processed small molecule organic solar cells. Sci. Rep. 5, 11143; doi: 10.1038/srep11143 (2015).


The E. coli mutant with ackA-pta, poxB, ldhA, and adhE deletions accumulated 0.67 g/L P3HB, up to 78.84% of cell dry weight in tube cultivation. The deletion of pyruvate formate-lyase gene pflB drastically decreased P3HB production and P3HB content to 0.09 g/L and 24.44%, respectively. Overexpressing pflB via the plasmid in its knocked out mutant restored cell growth and P3HB accumulation, indicating the importance of the pyruvate formate-lyase in microaerobic carbon metabolism. The engineered E. coli BWapld (pWYC09) produced 5.00 g/L P3HB from 16.50 g/L glucose in 24 h batch fermentation, and P3HB production yield from glucose was 0.30 g/g, which reached up to 63% of maximal theoretical yield.The alcohol dehydrogenase promoter P Thus, microbial production of P3HB via fermentation has attracted extensive attentions during the past few decades [Poly(3-hydroxybutyrate) (P3HB) is a polyester of 3-hydroxybutyrate that is synthesized by a variety of microorganisms as intracellular carbon and energy storages under unbalanced nutrients conditions . The mecAlthough considerable efforts have been devoted to decreasing the production cost of P3HB to make it economically competitive, the present price of P3HB is still not feasible to replace traditional petro-based plastics. Generally, P3HB was mainly produced from renewable carbon sources through aerobic fermentation , 5. Give phaCAB from Ralstonia eutropha was changed to the anaerobic alcohol dehydrogenase promoter PadhE, which was reported to be upregulated under oxygen limited condition. As a result, P3HB content was improved from 30% to 48% of cell dry weight [ E. coli undertakes mixed acid fermentation pathway, producing lactate, succinate, acetate, formate, and ethanol under oxygen limited conditions, which decreases the carbon flux into P3HB accumulation. E. coli mutant with deletions of ackA-pta, poxB, ldhA, and adhE was constructed and doubled the cell dry weight and improved P3HB production by 3.5-folds compared to the control [To achieve high P3HB content under microaerobic condition, the native promoter of P3HB biosynthesis operony weight . Moreove control .adhE promoter to drive the expression of phaCAB operon in E. coli mutants with defected mixed acid fermentation pathway. Moreover, the pyruvate formate-lyase, which catalyzes the coenzyme A-dependent, nonoxidative cleavage of pyruvate to acetyl-CoA and formate under anaerobic or microaerobic conditions, was overexpressed to investigate its effect on cell growth and P3HB accumulation  medium supplemented with 10 g/L glucose and then maintained at 37°C for 48 h as static cultures. For 5.5 liter bioreactor cultivation, 50 mL seed culture was transferred to the bioreactor containing 3 L LB medium supplemented with 20 g/L glucose. The pH was maintained at 7.0 via automatic addition of 5 M sodium hydroxide solution. Batch cultures were performed without air supply; the agitation was set at 75 rpm to prevent biomass sedimentation and maintain heat transfer and substrate exchange.2SO4 and then assayed with a gas chromatograph (GC) to measure the P3HB content. For glucose and by-products lactate, succinate, acetate, formate, and ethanol measurements, the supernatant of cell cultures was filtered through a 0.2 μm syringe filter and then analyzed by high-performance liquid chromatography (HPLC) equipped with an ion exchange column  and a refractive index detector.Cells were harvested by centrifugation at 8,000 ×g and 4°C for 10 min. The cell pellets were washed twice with distilled water and then lyophilized for 12 h for cell dry weight (CDW) assay. Lyophilized cells were subjected to methanolysis at 100°C for 4 h in the presence of 3% (v/v) HadhE has been demonstrated to improve the expression level of phaCAB operon under microaerobic condition [adhE controlled phaCAB was transformed into the wild type E. coli and a series of mixed acid fermentation mutants BWa, BWap, BWapl, BWapld, and BWapldf  and BWapld (pWYC09 + pMCS2pflB) were cultivated in oxygen limited 5.5 liter bioreactor to investigate cell growth and P3HB production profile. After 24 h cultivation, the recombinant BWapld (pWYC09) consumed 16.50 g/L glucose and produced 5.00 g/L P3HB; the P3HB content reached 73.58% of cell dry weight. The by-product formation was maintained at low level during the process, and finally 0.23 g/L succinate, 0.86 g/L acetate, 0.17 g/L formate, and 0.03 g/L ethanol were detected ( pflB was overexpressed, the final CDW and P3HB content were 5.88 g/L and 84.43%, respectively, accompanied with a total amount of 1.72 g/L mixed acid ( pflB helped to further improve P3HB content to 84.43%, which will facilitate downstream biopolymer purification processes.The recombinantdetected . When pfxed acid . Previou pta mutant JW2294 generated 16.60 g/L mixed acids ( pta mutant showed a 70% increase to 0.17 g/g. BWapld mutant reached up to 0.30 g/g, which was 2 times higher than that obtained from the wild type ( arcA mutant showed 46% of maximal yield under optimized microaerobic fermentation condition [ E. coli constructed here showed superior ability for microaerobic P3HB production compared to previous strains. Further optimization of different degrees of oxygen limitation in bioreactor batch cultivations should be performed to obtain much higher P3HB production titer and productivity. Since the overall redox balance is important to P3HB biosynthesis and the metabolic response [In terms of byproduct formation and P3HB production yield, the wild type strain BW25113 produced 13.60 g/L mixed acids, anded acids . In thisild type . The max pflB gene significantly decreased cell biomass and P3HB production and overexpressing pflB from the plasmid restored the cell growth and P3HB accumulation, indicating the importance of pyruvate formate-lyase for microaerobic metabolism. The engineered strain BWapld harboring pWYC09 accumulated 5.00 g/L P3HB, up to 73.58% of cell dry weight in 5.5 liter bioreactor fermentation. The P3HB production yield from glucose was 0.30 g/g, up to 63% of maximal theoretical yield. The optimization of phaCAB expression in mixed acid fermentation mutants was proved to be effective for improving microaerobically P3HB production.The alcohol dehydrogenase promoter and mixed acid fermentation mutants were combined to improve P3HB production under microaerobic condition. The deletion of


The free radical scavenging and antidiabetic activities of total anthocyanins from bitter melon (Momordica charantia Linn) fruit (TAMC) were evaluated by considering four harvesting times. The free radical scavenging activities of the TAMC samples were assessed using DPPH•, DMPD•+ and ABTS•+ assays against BHA, rutin and trolox standards. September as a harvesting period (TAMC-S) had effective DPPH• (SC50 2.55 ± 0.08 μg/mL), DMPD•+ (SC50 2.68 ± 0.09 μg/mL) and ABTS•+ (SC50 8.19 ± 0.09 μg/mL) scavenging activities compared with other samples and standards. In addition, August (TAMC-A) as a harvesting period showed very influential inhibitory activity against α-amylase (IC50 56.86 ± 1.12 μg/mL) and moderate inhibitory activity against α-glucosidase (IC50 88.19 ± 0.74 μg/mL). In comparison, pharmaceutical active ingredients such as acarbose exhibited anti-amylase and anti-glucosidase activities with IC50 values of 93.07 ± 1.49 μg/mL and 77.25 ± 1.20 μg/mL respectively. These results suggest that the correct selection of harvest period can significantly increase anthocyanin quantity because of the pharmaceutic properties of TAMC. Consequently, TAMC may be interesting for incorporation in pharmaceutical preparations for human health, since it can suppress hyperglycaemia that can be also used as food additives due to its antiradical activity.The majority of the antioxidant and antidiabetic activities of fruits are anthocyanins; a group of polyphenolics that are responsible for the color of many fruits, vegetables and flowers. The harvesting time, storage conditions, maturity, extraction steps  Anthocyanins are regarded as important nutraceuticals because of their antioxidant effects, which give them a potential role in prevention of the various diseases associated with oxidative stress , hydroxyl radicals (HO•) and non-free radical species such as H2O2 and singlet oxygen (1O2), are form of activated oxygen , which comprise free radicals like superoxide anion radicals  and type II noninsulin-dependent diabetes mellitus (NIDDM) are common and serious metabolic disorders throughout the world. Traditional plant treatments have been used in the world for the therapy of diabetes mellitus. Among many medications and other alternative medicines, several herbs have been known to cure and control diabetes; additionally they have no side effects. Plant-based medicine has been used cost-effectively worldwide to treat diabetes mellitus. In fact, in many parts of the world, this may be the only form of therapy available to treat diabetic patients. There are several reviews by different authors about anti-diabetic herbal plants , 12.Momordica charantia Linn. (Cucurbitaceae) commonly known as bitter melon, balsam apple, bitter gourd and bitter squash is a multi-purpose herb widely cultivated in many tropical and subtropical regions of the world. It is locally named kudretnarı and papara in Turkey. M. charantia fruits are used as vegetable in various region of the world. Apart from their role in food consumption, a wide array of pharmacological activities of M. charantia fruits such as antihyperglycemic, antidiabetic, antiulcer, antifungal, protein synthesis inhibitory activity, anti-tumor and antioxidant effects have been reported. M. charantia fruits contain many bioactive chemicals as flavonoids, saponions, peptides, lectins, triterpenoids, phenolic compounds , p-nitrophenyl-α-D-glucopyranoside, starch, α-amylase, α-glucosidase, N, N-dimethyl-p-phenylendiamine (DMPD•+), 2, 2ꞌ-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS•+), 3, 5-dinitrosalicylic acid (DNS), butylated hydroxyanisole (BHA), rutin hydrate, trolox, potassium persulfate and potassium sodium tartrate tetra hydrate were purchased from Sigma . All other chemicals were of analytical grade and obtained from either Sigma-Aldrich or Merck.Sodium chloride, ferric chloride, sodium hydroxide, sodium carbonate and sodium acetate were purchased from E. Merck . Acarbose, anhydrous ethanol, anhydrous dichloromethane, anhydrous ethyl acetate, glacial acetic acid, 2, 2-diphenyl-1-picryl-hydrazyl  was added onto the fruit materials (150 g) at a rate of 1: 15, which yielded to 2250 mL of solution. The extraction process has been continued during 2 h at a room temperature, using magnetic blender. Extract was filtered by the paper filter and the received solution was 1800 mL. Solution was treated with dichloromethane and ethyl acetate four times (250 mL x 4) for each, respectively. The remaining solution, approximately 1000 mL, was dried in the lyophilizator (Christ Alpha 1–2 LD Plus) at 10 μm Hg pressure at -50˚C. Finally, the residues were placed in a plastic flask and then kept at -30˚C until used.Determination of Antidiabetic ActivityAssay of α-Amylase InhibitionIn-vitro α-amylase inhibition was analyzed by following the method of Bernfeld (50 (the concentration required to inhibition of α-amylase activity by 50%). The IC50 values were determined by linear regression analysis using four different concentrations in triplicate and represent mean of the data. Individual blanks were prepared for correcting the background absorbance. In this case, the color reagent solution was added prior to the addition of starch solution and then the tube placed into the water bath. The other procedures were carried out as above. Controls were conducted in an identical fashion replacing TAMC with PBS (1.0 mL). Acarbose solution was used as positive control.Bernfeld  with minAssay of α-Glucosidase Inhibitionp-nitrophenyl-α-D-glucopyranoside (5.0mM) in the PBS was used as a substrate solution. TAMC samples and acarbose , dissolved at various concentrations in PBS, were mixed with enzyme solution and incubated during 15 min at 37˚C. Substrate solution (20 µL) was added and incubated during 15 min. The reaction was terminated by adding sodium carbonate solution  and absorbance was measured at 405 nm using spectrophotometer. The IC50 values of samples for the α-glucosidase inhibition activities were determined by linear regression analysis using four different concentrations in triplicate and represent mean of the data.A previously described bioassay method with minor modifications was used for measurement α-glucosidase inhibition of samples . The enzDetermination of Free Radical Scavenging ActivitiesDPPHRadical Scavenging Activity Assay• solution  in ethanol. The mixtures were shook forcefully and allowed to stand at room temperature in the dark during 30 min. Then, absorbance was recorded at 517 nm in a spectrophotometer. The results were expressed as SC50  by linear regression analysis.The DPPH radical scavenging abilities of samples were performed according to method of Blois  with minDMPD•+ Radical Scavenging Activity Assay•+ described by Fogliano et al. (•+ solutions (100 mM) was prepared in a deionized water. This solution (1 mL) was added to acetate buffer  and the colored radical cation (DMPD•+) was obtained by adding 0.2 mL of a of ferric chloride solution (0.05 M) . This solution (225 μL) was directly transferred to the tube and its absorbance was measured at 505 nm (absorbance of control tube). Different concentrations of TAMC samples or standards  and DMPD•+ (210 μL) were added to all tubes. Then, all tubes were stirred and left to stand for 10 min. After this time, a decrease in absorbance was measured at 505 nm in a spectrophotometer (absorbance of samples or standards). The buffer solution was used as a blank sample. The results were expressed as SC50 by linear regression analysis using four different concentrations in triplicate and represent mean of the data.Principal of the assay is based on reduction of the purple-colored radical DMPDo et al. . DMPD•+ ABTS•+ Radical Scavenging Activity Assay•+ radical cation scavenging capacity of TAMC samples and standards was examined according to chemical methods described by Re et al.  to give an absorbance of 0.750 ± 0.020 at 734 nm in 1 cm cuvette and all the assays were performed by equilibrating at 30˚C temperature. Then, the diluted ABTS•+ solution (1.0 mL) was added to TAMC samples or standards (3.0 mL) solution in PBS at different concentrations (2.5–15 µg/mL). The percentage inhibition of ABTS•+ was calculated for each concentration relative to a blank absorbance. The results were expressed as SC50 by linear regression analysis using four different concentrations in triplicate and represent mean of the data.ABTSe et al.  with slirsulfate . The reaDetermination of Total Anthocyanin ContentsThe total anthocyanin contents were carried out according to pH differential method described by Fuleki and Francis . The dripH 1.0 buffer : 1.86 g KCl was dissolved in 980 mL double-distilled water in a baker and the pH was adjusted to 1.0 ± 0.05 with HCl. The solution was transferred into 1 L volumetric flask and diluted to the volume with double-distilled water.pH 4.5 buffer : 54.43 g CH3COONa.3H2O was dissolved in 960 mL double-distilled water in a baker and the pH was adjusted 4.5 ± 0.05 with HCl. The solution was transferred into 1 L volumetric flask and diluted to the volume with double-distilled water.Total anthocyanin contents in the extracts determined as mg/L of cyanidin-3-glucoside (cyd-3-glu) equivalent using the following equation:A: (A520nm – A700nm) pH 1.0 – (A520nm – A700nm) pH 4.5, MWcyd-3-glu (molecular weight of cyanidin-3-glucoside): 449.2 g/mol, DF: dilution factor, l: path length in cm, ε = 26900 molar extinction coefficient for cyd-3-glu (L x mol–1 x cm–1) and 103: factor for conversion from g to mg.Where Statistical analysisExperimental results were given as mean ± SD of the three parallel measurements. Analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Duncan’s Multiple Range tests. p-values of < 0.05 were regarded as significant and P-values of < 0.01 very significant. Both operations were done with SPSS 15.0 for windows.Total Anthocyanin ContentsAnthocyanins and other flavonoids are regarded as an important nutraceuticals mainly due to their antioxidant effects. Moreover, they have also been used to modulate the activity of a wide range of enzymes and cell receptors. Regular consumption of anthocyanins and other polyphenols in fruits, vegetables, wines, jams and preserves is associated with probable reduced risks of chronic diseases as cancer, cardiovascular diseases, virus inhibition and Alzheimer’s disease . Total aα-Amylase Inhibition Activity50 value of 93.07 ± 1.49 μg/mL. The IC50 value of TAMC-A, TAMC-S, TAMC-O and TAMC-N were found to be 56.86 ± 1.12, 63.81 ± 0.86, 66.18 ± 1.34, 71.62 ± 1.01 μg/mL, respectively (p < 0.05). According to obtained results, TAMC showed appreciable α-amylase inhibitory effects when compared with acarbose. The author also suggests that M. charantia has high antidiabetic potential due to the total anthocyanins in it.Recently, natural sources of α-amylase inhibitor have received a lot of interest due to the side or mild effects as of synthetic enzyme inhibitors such as acarbose, metformin and orlistat and at the same time synthetic enzyme inhibitors can cause gastrointestinal distress . Certainα-Glucosidase Inhibition Activity50 value of 77.25 ± 1.20 μg/mL. The IC50 values of TAMC-A, TAMC-S, TAMC-O and TAMC-N were found to be 88.19 ± 0.74, 97.99 ± 1.40, 100.55 ± 1.73, 107.68 ± 0.98 μg/mL, respectively (p < 0.05). These results show that total athocyanins of M. charantia may be potential α-glucosidase inhibitor for diabetic disorder.α-glucosidase inhibitors such as acarbose, miglitol and voglibose are widely used in the treatment of patients with type II diabetes. α-glucosidase inhibitors delay the absorption of carbohydrates from the small intestine and thus they have a lowering effect on postprandial blood glucose and insulin levels . EarlierDPPH• Radical Scavenging Activity• radical scavenging is due to their hydrogen or electron donating abilities. DPPH•, a stable free radical, is transformed to stable diamagnetic molecule by receiving an electron or a hydrogen radical  of TAMC samples and standards on the DPPH• increased in that order: TAMC-S > (2.55 ± 0.08) > TAMC-O (3.13 ± 0.02) > TAMC-A (4.93 ± 0.24) > TAMC-N (7.39 ± 0.10) > BHA (8.94 ± 0.21) > rutin (17.47 ± 0.17) > trolox (25.74 ± 0.46) (p < 0.05)  .DMPD•+ Radical Scavenging Activity•+ radical cation solution becomes lighter and absorbance of solution decreases in the presence of an antioxidant compound. The DMPD•+ radical cation solution shows a maximum absorbance at 505 nm. Antioxidant compounds which are hydrogen donors to DMPD•+ quench the color of DMPD•+ solution   .ABTS•+ Radical Scavenging Activity•+ has been extensively used to evaluate the antioxidant capacity of complex mixtures and individual compounds. The reaction of the preformed radical with free-radical scavengers can be easily monitored by following the decay of the sample absorbance at 734 nm. The ABTS•+ radical cation can be prepared employing different oxidants. Results obtained using a potassium persulfate as oxidant show that the presence of peroxodisulfate increases the rate of ABTS•+ autobleaching in a concentration-dependent manner. ABTS•+ radical cation was generated in the ABTS/K2S2O8 system S4•- +ABTS →SO4-2 + ABTS•+                    (2)SO2O8-2 + 2ABTS →2SO4-2 + 2ABTS•+S•+ radical cation forms the basis on one of the spectrophotometric methods that have been applied to the measurement of the total antioxidant activity of pure substances, solutions, aqueous mixtures and beverages  < TAMC-S (8.19 ± 0.09) < BHA (8.47 ± 0.19) < TAMC-O (8.56 ± 0.02) < TAMC-A (9.31 ± 0.16) < TAMC-N (9.62 ± 0.15) < rutin (15.68 ± 0.39) (p < 0.01) (Generation of the ABTSeverages . For rad < 0.01) .R = 0.984, p < 0.05). Similarly, those between anthocyanin contents and DMPD radical scavenging activity , ABTS radical scavenging activity  were significant. Additionally, the correlation between total anthocyanin contents and α-amylase inhibition activity , α-glucosidase inhibition activity  were significant.The correlation coefficient between DPPH radical scavenging activity and total anthocyanin contents was statistically significant (i.e. diabetes, asthma, eczema, premenstrual syndrome, rheumatoid arthritis, migraine, menopausal symptoms, etc. and can be used for maintaining general health. A lot of medicinal plants in the literature have hypoglycemic and other beneficial properties. When they are correctly used, medicinal plants are considered safer than conventional medications. People are greatly concerned about the efficacy and side effects of many synthetic drugs and hence they choose herbal medicines for providing a safe and natural alternative treatment for many health problems. In fact, the herbs are always the alternative medicine and primary source. The advantages of using medicinal plants are numerous. Otherwise, they typically have fewer side effects and may be safer to use over time. M. charantia fruits may be useful for the treatment of antidiabetic by reduction the α-amylase and α-glucosidase enzymes activities due to the presence of anthocyanin contents in it. In addition to this TAMC samples demonstrated very effective scavenging activities depend on the DPPH•, DMPD•+ and ABTS•+ radicals but these properties change by depending on harvest times. When these activities compared with the synthetic antioxidants, they are very important for regulation of oxidative stress owing to theirs side effects. By this way, the obtained results in this study show that total anthocyanins of M. charantia fruits can be used as easy accessible source of natural antidiabetics and antioxidants as a possible food supplement or in a pharmaceutical and medical industry. For this reason, it could be performed on the isolation and characterization of the anthocyanin compounds from TAMC and to evaluate its in-vivo effects in next works.History showed that medicinal plants have been used in traditional healing around the world for a long time for the treatment of many diseases


Background. This study was to explore the pharmacokinetics of saxagliptin (Sax) in Goto–Kakizaki (GK) rats complicated with depression induced by chronic unpredicted mild stress (CUMS). The comorbidity of diabetic patients with depression is becoming more and more epidemic. Whether depression mental disorder alters the pharmacokinetics of hypoglycemic drugs in diabetes patients is not clear.Methods. Five-week-old male GK rats were kept in the cage for 7 weeks in a specific pathogen free (SPF)-grade lab until the emergence of diabetes and were then divided into two groups: control group and depression model group. Rats in the CUMS-induced depression group were exposed to a series of stressors for 8 weeks. Plasma serotonin and dopamine levels and behavior of open-field test were used to confirm the establishment of the depression model. All rats were given 0.5 mg/kg Sax orally after 8 weeks and blood samples were collected at different time points. The Sax concentration was assayed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The CYP450 activity of the liver microsomes was determined by using cocktails of probe drugs in which the activities of CYP enzymes were assessed through the determination of the production of the probe drugs.Results. Statistically significant differences in Sax pharmacokinetics were observed for area under curve, clearance, peak concentration, peak time and mean residence time between the depression rats and the control rats, while no statistical differences were observed for half-time and distribution volume by HPLC-MS/MS analysis. The CYP450 activity had different changes in the depression group.Conclusions. These results indicated that CUMS-induced depression alters the drug metabolic process of Sax and CYP450 activity of the liver microsomal enzymes in GK rats. Depression mental disorder is increasing in patients with obesity, hypertension, myocardial infarction, and diabetes . Type 2 In recent years, many researchers use chronic unpredicted mild stress (CUMS)-induced depression to simulate animal models of depression. Rats are exposed to a variety of mild stressors sequentially over a period of weeks in the CUMS model. These chronic unpredicted mild stressors are the major causes of depression .β-cells and inhibit the hepatic glucagon production of α-cells  is one of the DPP-4 inhibitors which is more safe and tolerable  inhibitors are used widely as a brand new type of oral hypoglycemic drug for treating T2DM , which d α-cells . Saxagliolerable . Sax exholerable . It neitolerable .Sax is chiefly metabolized by the hepatic microsomal enzyme CYP3A4, which is a vital part of the CYP450 family. The cytochrome P450s constitute the major drug-metabolizing enzyme system. CYP3A4, CYP2D6 and other isozymes, along with CYP2C, CYP1A2, CYP2E1, are the main human CYP isoforms catalyzing the metabolism of the vast majority of the commonly prescribed drugs. A previous work from the authors of this study found that CUMS-induced depression alters pharmacokinetics of mitiglinide in Sprague–Dawley rats . This stSax standard sample and sitagliptin internal standard (Sit) were purchased from TRC  and TLC , respectively. Phenacetin (Phe), paracetamol (Par), venlafaxine (Ven), desvenlafaxine (Dven), nifedipine (Nif), dehydronifedipine (Dnif) standard sample and loratadine internal standard (Lor) were purchased from Sigma–Aldrich (US). Sax medicine was obtained from Sino–American Shanghai Squibb Pharmaceuticals Co., Ltd. High performance liquid chromatography (HPLC)-grade methanol and ethyl acetate were purchased from Merck (US).Male GK rats, 5 weeks old, purchased from Shanghai SLAC Laboratory Animal Co., Ltd., Animal Quality Certificate: 2007000562918, were kept in the Laboratory Animal Center, East China Normal University, Shanghai . Rats were kept in the cage for 7 weeks in an SPF-grade lab until the emergence of diabetes (with blood glucose level ≧ 11 mmol/L). Then the diabetic rats were randomly divided into two groups (each group comprising nine rats): control group and depression model group.ad libitum. The stressors included restraint , hot water swimming , cold water swimming , clip tail , cages tilting , horizontal shaking (10 min), damp padding (24 h), noise interference (10 min), and day/night inversion (24 h). Each stressor was used 5–6 times randomly  for 5 min.Before and after the model establishment, the open-field behavior of each rat was analyzed using a ZS-ZFT Video Analysis System . The apparatus was an opaque box (100 cm ×100 cm × 40 cm). The open-field area was divided into 33 ×33 cmg for 5 min. The plasma serotonin (5-HT) and dopamine (DA) levels were measured with enzyme-linked immunosorbent assay .Before and after the model establishment, 1.0 mL blood samples were collected from each rat by eye canthus and centrifuged to obtain 0.5 mL plasma at 3,000 × g for 5 min. The samples were stored at −80 °C for analysis.All rats were fasted overnight but with free access to water. 0.5 mg/kg Sax (suspended in 5 g/L Carboxymethyl cellulose-Na) was given orally. Blood samples were collected by eye canthus at 0.17, 0.33, 0.5, 0.83, 1.17, 1.5, 2, 3, 5, 8 h. Then plasma samples were centrifuged to obtain plasma at 3,000 × g for 3 min. The supernatant was evaporated to dry. The residue was re-dissolved with 100 µL methanol via vortex-mixed for 1 min and centrifuged at 16,000 × g for 1 min. 2.0 µL of the supernatant was injected into the HPLC-MS/MS system for analysis.The Sax concentration was assayed by the HPLC-tandem mass spectrometry (MS/MS) method . A total18 column . The mobile phase contained 35% methanol and 65% water (0.1% ammonium formate) with a flow rate 0.4 mL/min. The optimized conditions of MS were as follows: positive electrospray ionization (ESI+) mode, capillary at 4,000 V, ion-spray gas temperature at 350 °C, gas flow rate at 11 L/min, and nebulizer at 15 psi. The parameters of Sax and Sit were as follows: fragmentary voltage at 110 V and 130 V, collision energy at 22 units and 18 units, respectively. The multiple-reaction monitoring mode was selected for quantifying of Sax and Sit, for which the precursor-to-product ion transitions were 316.1 → 179.9 and 407.9 → 234.9, respectively. The MassHunter Workstation software  was used to collect and process data.The HPLC equipped with a reverse-phase column was connected to a triple quadrupole tandem MS with an electrospray interface. The system consists of the LC system  with a G1322A degasser, a G1318B auto-modifiable pump, a G1367E auto-sampler, a G1316A adjustable column temperature box, and the Agilent 6420 MS system. Chromatographic separation was achieved using a ZORBAX EP-Cg at 4 °C for 20 min, and the supernatant was further centrifuged at 105,000 × g at 4 °C for 60 min. The helvus sediment was reconstituted with 1.0 mL Tris–HCl buffer and stored at −80 °C for analysis.The control and CUMS-induced rats were fasted for 12 h and killed by cervical dislocation before removal of the liver. The liver was excised, rinsed with ice-cold normal saline , weighed for 1 g, and homogenized with 4.0 mL in a 0.05M Tris–HCl buffer (pH 7.4). The homogenate was centrifuged at 10,000 × The total protein concentrations of liver microsomal enzymes were determined by using a Bicinchoninic Acid Protein Assay Kit. The CYP450 activity of the liver microsomes was determined by using cocktails of probe drugs in which the activities of CYP enzymes were assessed through the determination of the production of the probe drugs. The Phe, Ven, Nif were metabolized to Par, Dven, Dnif by CYP1A2, CYP2D6, CYP3A4, respectively and these products were determined by HPLC-MS .g for 10 min at 4 °C. The supernatant was extracted into a new brown centrifuge tube and evaporated to dry at 80 °C. The residue was re-dissolved with 200 µL methanol via vortex-mixed for 1 min and centrifuged at 15,000 × g for 2 min. 5.0 µL of the supernatant was injected into the LC-MS/MS system for analysis.The incubation mixtures  contained 1 mg/mL microsomal protein, 0.1 M phosphate buffer (PH = 7.4), 1 mM nicotinamide adenine dinucleotide phosphate (NADPH), probe drugs . A total of 100 µL microsomal protein and 100 µL probe drugs were added with 250 µL phosphate buffer in a brown centrifuge tube, vortex-mixed for 1 min, and incubated at 37 °C water. After 5 min pre-incubation, the reactions were initiated by the addition of 50 µL NADPH. The incubations were performed at 37 °C for 20 min in a water bath and terminated by adding 1.4 mL cold ethyl acetate and 20 µL Lor (5 µg/mL). The samples were vortex-mixed for 2 min and centrifuged at 3,500 × 18 column . The mobile phase contained of methanol (A) and water  with a flow rate of 0.5 mL/min. The gradient elution were 70% A (0–1 min), 80% A (1–2 min), and 90% A (2–7.5 min). The optimized conditions of MS were as follows: ESI+ mode, capillary at 3,500 V, ion-spray gas temperature at 350 °C, gas flow rate at 10 L/min, and nebulizer at 15 psi. The parameters of Par, Dven, Dnif and Lor were as follows: fragmentary voltage at 150 V, 100 V, 80 V and 140 V, and collision energy at 30 units, 18 units, 14 units and 22 units, respectively. The monitoring ions were set as m/z 345.3 → 284.1 for Dnif, 264.3 → 58.2 for Dven, 152.1 → 110 for Par and 383.8 → 338.1 for Lor.Chromatographic separation was achieved using a ZORBAX XDB-Ct Test. Data was expressed as mean ± standard deviation (SD). p < 0.05 was considered statistically significant.Statistical analysis and pharmacokinetics parameter analysis were performed with SPSS 16.0  and DAS 2.1 software . Differences between the two groups were analyzed via independent-samples The animal experiment protocol was approved by the Bioethics Committee of East China Normal University .a, b) and plasma levels  were same between the two groups before the depression model establishment, but significantly changed after the depression model establishment , and from 14.10 ± 2.38 to 7.60 ± 2.17 (p < 0.01) in the depression group, respectively. However, no change occurred for these scores in the control group (p > 0.05). After the model establishment, significant decreases were seen for these scores between the two groups .The behavior scores of open-field test and 5-HT and DA plasma levels are shown in p < 0.01) and from 0.55 ± 0.07 to 0.21 ± 0.05 (p < 0.01) within the depression group, respectively. No significant alteration was reported within the control group (p > 0.05). A notable difference was also observed between the two groups at the end of model establishment .5-HT and DA plasma levels were also decreased from 62.09 ± 12.76 to 22.17 ± 4.28 (R2 > 0.99) by weight of 1∕x2 over the concentration range of 1.25–50 ng/mL. The lowest limit of quantitation (LLOQ) was 1.25 ng/mL. The matrix effects were approximately 96%. Accuracy and intra-day precision, and inter-day precisions were 89.22%–106.70%, 1.12%–3.51% and 0.73%–2.89%, respectively , peak time (Tmax), peak concentration (Cmax) and mean residence time (MRTt0−) in the depression group significantly decreased by 33% (from 67.06 ± 14.12 to 44.63 ± 15.49), 29% (from 1.46 ± 0.38 to 1.04 ± 0.25), 27% (from 20.48 ± 4.61 to 14.85 ± 6.03) and 24% (from 2.55 ± 0.32 to 1.95 ± 0.57), respectively (p < 0.05). The distribution volume (Vz/F) and clearance (CLz/F) increased, but only CLz/F had a notable difference (p < 0.05). No statistically significant difference was observed in half-time (Tz1∕2) between the two groups.The concentration-time parameters were calculated with noncompartmental analysis method . The areR2 > 0.99). The average accuracy of three QC level samples of the Par, Dven, Dnif were 106.77%, 98.87% and 103.55%, respectively. The average intra-day precision and inter-day precision of three products were 5.89%, 2.50%, 2.23% and 8.15%, 4.80%, 4.07%, respectively. The average extraction recoveries of three products and Lor were 90.32%, 89.68%, 89.88% and 90.19%, respectively. The average matrix effects and LLOQ of Par, Dven, Dnif were 91.76%, 90.97%, 88.69% and 7.0, 1.0, 10.0 ng/mL, respectively . The CYP2D6 and CYP1A2 activity also varied statistically, but the varying degrees were not very obvious between the two groups. The CYP2D6 activity increased from 0.1713 ± 0.0122 to 0.1914 ± 0.0172 ng/(min mg protein) (p < 0.05). However, the CYP1A2 activity decreased from 0.1770 ± 0.0053 to 0.1688 ± 0.0079 ng/(min mg protein) (p < 0.05)  ( < 0.05) .in vivo and the outward appearance respectively. This study showed that the behavior scores of open-field test and 5-HT and DA plasma levels had verified the successful establishment of the depression model induced by CUMS. Moreover, the 5-HT plasma levels and exploratory scores had strong positive correlation in the depression group (r = 0.708). There was also a strong positive correlation between DA plasma levels and locomotion scores in the control group (r = 0.792). The results showed a significant correlation exist between the plasma levels of these two monoamines and the change of behavior in the open-field test. Thus, the GK rats of depression group could better imitate the diabetic patients complicated with depression, and the control group resembled diabetes. To keep the reproducibility and consistency in the depression model establishment, the stressor sequences in the lab were generally the same for all the experiments.Chronic unpredictable mild stress (CUMS) has been widely used for many year in the research of depression . The behThe previous work had found that CUMS-induced depression could significantly increase the activity of the hepatic drug-metabolizing enzyme CYP450 , and phain vivo, we detected metabolism of Sax using liver microsomal enzyme in vitro with cocktails method. The results showed that, in the control group, the concentration of Sax decreased from 0.50 to 0.35 mg/mL in the control group, while in the depression group it was significantly decreased from 0.50 to 0.15 mg/mL. It indicated that CUMS-induced depression GK rats could alter the metabolic process of Sax by increasing the activity of the hepatic drug-metabolizing enzyme CYP3A4. Besides, the CUMS-induced depression GK rats changed not only CYP3A4 activity, but also CYP2D6 and CYP1A2 activity. Other researchers revealed that the genotype and phenotype of CYP450s had individual differences (It is well known that Sax is mainly metabolized by cytochrome 3A enzyme in the liver. The main CYP3A isoforms, CYP3A4, CYP3A5 and CYP3A7, constitute almost 60% of total P450 content . The actferences . So the ferences . These hferences .These results indicated that CUMS-induced depression alters the drug metabolic process of Sax and CYP450 activity of the liver microsomal enzymes in GK rats. These data can provide some basic evidence for clinical rational use of drugs. From the results of this work in GK rats and our previous works in SD rats, we can deduce that chronic unpredicted mild stress-induced depression alter the liver metabolic enzyme activity, and then cause the changing of pharmacokinetics. This phenomenon reminds that depression patients may be also subjected to the same situation. Therefore, therapeutic regimen should be largely ameliorated in clinical practice for depression patients.10.7717/peerj.1611/supp-1Supplemental Information 1The weight of GK rats from 8 weeks to 22 weeks between the two groups.Click here for additional data file.10.7717/peerj.1611/supp-2Supplemental Information 2Open-field test: locomotion score and exploratory score, beofe and after model establishment. 5-HT and DA plasma levels. CYP3A4, CYP2D6 and CYP1A2 activity of liver microsomal enzymes.Click here for additional data file.


Ethyl pyruvate (EP) is a simple aliphatic ester of the metabolic intermediate pyruvate that has been demonstrated to be a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems. However, the protective effects and mechanisms underlying the actions of EP against endothelial cell (EC) inflammatory injury are not fully understood. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of EC inflammation. In this study, our aim was to explore the effects of EP on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human umbilical vein endothelial cells (HUVECs) and to explore the role of ERS in this process. TNF-α treatment not only significantly increased the adhesion of monocytes to HUVECs and inflammatory cytokine  production in cell culture supernatants but it also increased ICAM and MMP9 protein expression in HUVECs. TNF-α also effectively increased the ERS-related molecules in HUVECs . EP treatment effectively reversed the effects of the TNF-α-induced adhesion of monocytes on HUVECs, inflammatory cytokines and ERS-related molecules. Furthermore, thapsigargin  attenuated the protective effects of EP against TNF-α-induced inflammatory injury and ERS. The PERK siRNA treatment not only inhibited ERS-related molecules but also mimicked the protective effects of EP to decrease TNF-α-induced inflammatory injury. In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS. The incidence of cardiovascular diseases, such as atherosclerosis (AS), is increasing globally and has become a costly public health issue Pyruvate, which is the anionic form of a simple alpha-keto acid, plays a key role in intermediary metabolism as a product of glycolysis and as the starting substrate for the tricarboxylic acid (TCA) cycle 2+ imbalances or perturbations in protein glycosylation, leading to the accumulation of unfolded proteins in the ER and the activation of the unfolded protein response (UPR) pathway The endoplasmic reticulum (ER) is an organelle involved in protein folding and modification, and it acts as a major intracellular calcium store Most importantly, we assessed the anti-inflammatory effects of EP in human umbilical vein endothelial cells (HUVECs) and explored the role of ERS in this study.EP, thapsigargin , tumor necrosis factor-α (TNF-α), dimethyl sulfoxide (DMSO) and the antibody against TNF-α were purchased from the Sigma-Aldrich Company . The Cell Counting Kit-8 (CCK8) was purchased from Dojindo . PERK siRNA and the antibodies against GRP78, intercellular cell adhesion molecule 1 (ICAM-1), matrix metalloproteinase 9 (MMP9) and caspase12 were obtained from Santa Cruz Biotechnology . Human soluble adhesion molecule ICAM-1 (sICAM-1), soluble E-selectin (sE-selectin), interleukin (IL-8) and monocyte chemoattractant protein-1 (MCP-1) ELISA kits were obtained from R&D Systems . The antibodies against phosphorylated-PERK (p-PERK), PERK, ATF4 and β-actin were obtained from Cell Signaling Technology . The rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company .2 and 95% air. The EP solution was prepared in DMSO and diluted with culture medium immediately prior to the experiment. DMSO (0.01%) was used as the control group. As described in previous studies HUVECs  were cultured in RPMI 1640 medium  supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin at 37°C in 5% COCCK-8 was used to measure cell viability. Cells were cultured in a 96-well plate and exposed to various treatments, according to the manufacturer's protocols. The control group was treated with 0.1% DMSO. Then, 10 µl of CCK-8 was added to each well, and the plate was incubated at 37°C for 2 h. Optical density (OD) values were assessed at 450 nm using a microplate reader , and cell viability was expressed in terms of the OD value.4/ml DMEM medium containing 1% FBS) were then added. After incubation for 1 h, the HUVEC monolayer was gently washed with phosphate-buffered saline (PBS) to remove unbound monocytes. The fluorescence was measured to determine the levels of bound monocytes using a microplate reader  at excitation and emission wavelengths of 496 and 520 nm, respectively.Monocyte adhesion to ECs was determined using U937 cells  as previously described by our group After treatment, the cell culture supernatants were collected, and the production of sICAM1, sE-selectin, MCP-1 and IL-8 by the HUVECs was measured using special ELISA kits.For siRNA transfections, HUVECs were plated onto 6-well, 24-well or 96-well plates and allowed to grow to subconfluency. The cells were transiently transfected with the negative control or PERK siRNA at 100 pM for 24 h using the Lipofectamine RNAiMAX reagent  in OPTI-MEM media . The cells were subsequently prepared for use in further experiments.After treatment, the HUVECs were lysed in sample buffer , sonicated, boiled, run through an 8–12% Bis/Tris gel using 5× MES buffer (Invitrogen) and transferred to an Immobilon NC membrane . The membranes were blocked with 5% nonfat milk in TBST  and then probed with antibodies against GRP78, ICAM1, caspase12 and MMP9 (1∶500) and against p-PERK, TNF-α, ATF4 and β-actin (1∶1000) overnight at 4°C. The membranes were then placed in blocking buffer, washed with TBST, probed with secondary antibodies (1∶5000) in blocking buffer at room temperature for 90 min and washed. Fluorescence was detected using a Bio-Rad imaging system , and the signals were quantified using the Image Lab Software .t-test. A difference of P<0.05 was considered to be statistically significant. All of the experiments were repeated three times.All of the values are presented as the mean ± standard deviation (SD). Group comparisons were performed using ANOVA (SPSS 13.0). All of the groups were analyzed simultaneously using the LSD P>0.05). However, TNF-α increased sICAM-1 production in the cell culture supernatants to 6.14±0.41, 11.93±0.85 and 15.01±1.26 pg/well, for each respective trial . In addition, TNF-α increased the expression of the ERS-related protein GRP78, as well as ATF4, cleaved-caspase12 and p-PERK  for 6 h to explore the effects of TNF-α on cell viability, inflammatory cytokines and ERS-related molecules. As shown in P>0.05). Exposure of the HUVECs to TNF-α significantly increased their binding to the U937 monocytes to 874.02±51.69% . In addition, exposure of the HUVECs to TNF-α significantly increased the production of sICAM1, IL-8, MCP-1 and sE-selectin to 14.59±1.30 pg/well, 9.17±0.75 ng/well, 14.71±1.21 ng/well and 1.715±0.128 ng/well in the cell culture supernatants , and the effects were the most obvious at 10 mM EP, which led to decreased sICAM1, IL-8, MCP-1 and sE-selectin production to 6.95±0.57 pg/well, 4.15±0.41 ng/well, 6.94±0.65 ng/well and 0.810±0.064 ng/well. TNF-α (10 ng/mL) significantly up-regulated the expression of the ICAM-1 and MMP9 proteins to 496.15±32.87% and 320.47±19.95% in the HUVECs HUVECs were treated with EP  for 2 h and then subjected to TNF-α(10 ng/mL) treatment. As shown in P<0.01). EP treatment reversed the effects of TNF-α, decreasing the GRP78, cleaved-caspase12, ATF4 and p-PERK expression to 96.44±5.50%, 52.94±4.26%, 67.44±4.31% and 128.60±6.88% at 10 mM EP .As shown in P<0.01, P<0.01). In addition, EP treatment also decreased ICAM-1, MMP9 and TNF-α protein expression to 31.09±3.80%, 25.14±3.30% and 45.61±4.50% in the HUVECs . Compared with the TNF-α group, THA+TNF-α treatment not only significantly increased the binding of U937 monocytes to HUVECs and increased sICAM1 and MCP-1 production in the cell culture supernatants  for 2 h in the absence or presence of THA  and then subjected to TNF-α (10 ng/mL) treatment. EP treatment significantly inhibited the TNF-α-induced binding of U937 monocytes to the HUVECs to 39.81±4.05% and decreased the production of sICAM1 and MCP-1 in the cell culture supernatants to 6.80±0.57 pg/well and 7.03±0.60 ng/well , while THA and EP co-treatment reversed the down-regulation of GRP78, ATF4, cleaved-caspase12, and p-PERK protein expression to 78.07±4.35%, 32.29±3.75%, 24.80±3.48% and 138.14±7.02% . Compared with the TNF-α group, THA+TNF-α treatment significantly increased GRP78, ATF4, cleaved-caspase12 and p-PERK protein expression to 168.42±8.90%, 167.14±9.01%, 269.66±15.89% and 205.60±11.87% in the HUVECs .As shown in P<0.01, P<0.01, P>0.05, P>0.05, To further explore the role of ERS in the anti-inflammatory effects, HUVECs were treated with PERK siRNA for 24 h and then subjected to TNF-α (10 ng/mL) treatment. PERK siRNA treatment significantly inhibited the TNF-α-induced binding of U937 monocytes to HUVECs to 619.47±36.92% and decreased sICAM1 and MCP-1 production in the cell culture supernatants to 8.41±0.66 pg/well and 8.52±0.71 ng/well , while PERK siRNA+TNF-α treatment significantly decreased GRP78, ATF4, cleaved-caspase12 and p-PERK protein expression to 191.28±10.37%, 235.71±14.02%, 293.20±16.07% and 125.09±7.80% . Compared with the control siRNA group, PERK siRNA treatment alone significantly decreased GRP78 and p-PERK protein expression to 41.52±4.70%, 82.95±5.27%, 70.28±4.81% and 22.13±3.46% .As shown in Although the pathogenesis of atherosclerotic vascular disease involves multifactorial processes, accumulating evidence demonstrates that inflammation and subsequent endothelial dysfunction play fundamental roles in the initiation and progression of atherosclerosis Accumulating evidence suggests that the inflammatory cytokine TNF-α, which is a pleiotropic proinflammatory cytokine, plays an important role in the disruption of vascular function and the subsequent development of vascular disease EP has been demonstrated to protect against the inflammatory response in some cardiovascular diseases. For example, it has the ability to inhibit neutrophil activation, inflammatory cytokine release, and nuclear factor κB translocation. EP has been associated with a delayed myocardial protective effect after regional ischemia and reperfusion (IR) injury in an in vivo rat heart model Previous studies have confirmed that ERS plays an important role in regulating the pathological process of EC inflammation. Shinozaki and colleagues have confirmed that a deficiency in Herp (an ERS-related protein) suppresses atherosclerosis in apolipoprotein E knockout mice by attenuating the inflammatory response In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS. Furthermore, the inhibition of the ERS pathway confers a protective effect against endothelial inflammatory injury, indicating that ERS is a crucial mediator of endothelial inflammation.