Background. The objective of the present study was to perform a bioguided fractionation of unripe Rubus coreanus Miquel (uRC) and evaluate the lipid accumulation system involvement in its antiobesity activity as well as study the uRC mechanism of action. Results. After the fractionation, the BuOH fraction of uRC (uRCB) was the most active fraction, suppressing the differentiation of 3T3-L1 adipocytes in a dose-dependent manner. Moreover, after an oral administration for 8 weeks in HFD-induced obese mice, uRCB (10 and 50 mg/kg/day) produced a significant decrease in body weight, food efficiency ratio, adipose tissue weight and LDL-cholesterol, serum glucose, TC, and TG levels. Similarly, uRCB significantly suppressed the elevated mRNA levels of PPARγ in the adipose tissue in vivo. Next, we investigated the antiobesity effects of ellagic acid, erycibelline, 5-hydroxy-2-pyridinemethanol, m-hydroxyphenylglycine, and 4-hydroxycoumarin isolated from uRCB. Without affecting cell viability, five bioactive compounds decreased the lipid accumulation in the 3T3-L1 cells and the mRNA expression levels of key adipogenic genes such as PPARγ, C/EBPα, SREBP-1c, ACC, and FAS. Conclusion. These results suggest that uRC and its five bioactive compounds may be a useful therapeutic agent for body weight control by downregulating adipogenesis and lipogenesis. A World Health Organization (WHO) expert committee proposed a classification of overweight and obesity that applies to both men and women and to all adult age groups , 2. Obesγ (PPARγ), sterol regulatory element binding protein-1c (SREBP-1c), CCATT/enhancer binding protein α (C/EBPα), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), which promotes adipogenesis [γ and C/EBPα induces adipocyte differentiation by activating the expression of adipocyte-specific proteins such as adiponectin, resistin, and apolipoprotein E, indicating that these transcription factors are important regulators of adipocyte differentiation [Lipid accumulation reflects the process of preadipocytes differentiating into adipocytes, and this process is regulated by the increased expression of various transcription factors and adipogenesis-related genes, such as peroxisome proliferator-activated receptor ogenesis . Increasntiation . At the ntiation , 9. Rubus coreanus Miquel, commonly called Bokbunja in Korea, is native to far-eastern Asian countries . R. coreanus has been used for centuries as a traditional alternative medicine. R. coreanus fruits are usually harvested at the red stage (ripe). When ripe, R. coreanus fruits are soft and differ from unripe fruits in color . Various studies have demonstrated that the ripe fruits of R. coreanus significantly reduce the risk of many diseases, including asthma, allergies, and obesity [ R. coreanus contains various bioactive compounds, including phenolic acids, triterpenosides, flavonoids, and ellagitannin [ R. coreanus is ellagic acid, which is in a significantly higher concentration in unripe than ripe R. coreanus [ in vitro and in vivo [ R. coreanus on antiobesity have not been investigated. Therefore, further studies will be required in order to define the bioactive compounds that regulate the antiobesity effects of R. coreanus.The fruit of obesity , 11, and obesity –14. It hgitannin . A majorcoreanus . Moreove in vivo . However R. coreanus alleviated fatigue by elevating the antioxidative potential [ R. coreanus (uRC) extract was higher than that of the ripe one in high fat diet- (HFD-) induced obese mice [We previously reported that ripeotential . Recentlese mice . These r R. coreanus (uRCB) on body weight, adipose tissue weight, serum lipid profiles, and the expression levels of the PPARγ gene associated with lipid metabolism on epididymal adipose tissue in high fat diet- (HFD-) induced obese mice were studied. In addition, to evaluate the effect of bioactive compounds of uRCB on the differentiation of 3T3-L1 adipocytes, we measured lipid accumulation by Oil Red O staining. The inhibitory effects of five candidates, (1) erycibelline, (2) 5-hydroxy-2-pyridinemethanol, (3) m-hydroxyphenylglycine, (4) ellagic acid, and (5) 4-hydroxycoumarin, were further confirmed by downregulation of the expression of PPARγ, C/EBPα, SREBP-1c, ACC, and FAS.In this context, our aim was to evaluate the involvement of the lipid accumulation system in the antiobesity effect of the active fraction and phytochemically characterize the extract and its active fraction. To evaluate the effects of five organic solvent fractions isolated from uRC on the differentiation of 3T3-L1 adipocytes, we measured lipid accumulation by Oil Red O staining. Moreover, the effects of the BuOH fraction obtained from unripe3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, 3--2,5-diphenyltetrazolium (MTT), Oil Red O, dimethyl sulfoxide (DMSO), m-hydroxyphenylglycine, p-aminophenol, ellagic acid, and 4-hydroxycoumarin were purchased from Sigma Chemical Co. . Erycibelline was purchased from ChemFaces . 5-Hydroxy-2-pyridinemethanol was purchased from IS Chemical Technology . Dulbecco's Modified Eagle's Medium (DMEM), bovine serum (BS), and fetal bovine serum (FBS) were purchased from Invitrogen Inc. . All other chemicals were analytical reagent grade. R. coreanus  fruits used in this study were collected (May 2013) in Gochang County  and authenticated by Dr. Kim at the Jeollanamdo Institute of Natural Resources Research (JINR), Jangheung, Jeollanamdo, South Korea. uRC (2.5 kg) was extracted using 20 volumes of 5% ethanol at 100°C for 4 h. The extracted solution was then filtered, concentrated with an evaporator under a vacuum, and freeze-dried. These dried 5% ethanol extracts of uRC were stored at 4°C to avoid compound degradation before they were used in the experiments. The dried 5% ethanol extract of uRC (30 g) was suspended in water and successively divided with n-hexane (3 × 500 mL), chloroform , ethyl acetate , and n-butanol .The unripe5 cells/mL in a six-well plate for 2 days until confluent (designated as day −4). Two days later (designated as day −2), the media were changed to DMEM with 10% BS. Next, two days after confluence (designated as day 0), the cells were cultured in DMEM 10% FBS and a differentiation cocktail (MDI) consisting of 10 μg/mL insulin, 0.5 mM IBMX, and 1 μM DEX, with or without a sample. After 2 days, the cells were cultured in DMEM containing 10% FBS and 10 μg/mL insulin, which was then changed to DMEM containing 10% FBS (designated as day 4). Two days later (designated as day 6), the media were changed to DMEM with 10% FBS. Orlistat (20 μM), and a known antiobesity drug, was used as the positive control [3T3-L1 preadipocytes were purchased from the ATCC  and cultured in DMEM supplemented with 10% BS. 3T3-L1 cells were seeded at a density of 1 × 10Orlistat  μM, and  control .Oil Red O staining was performed on day 8. The differentiated cells were washed with phosphate buffered saline (PBS) and then fixed in 10% formaldehyde for 30 min. The cells were then washed with distilled water. The fixed cells were stained with an Oil Red O working solution for 30 min and then washed 3 times with distilled water. Differentiation was monitored using microscopy and quantified via elution with 100% isopropanol. Optical density (OD) measurements were obtained at 500 nm.4 cells/well in a 96-well plate (n = 6 replicates) and allowed to adhere overnight. Then, various concentrations of five organic solvent fractions, six subfractions, and bioactive compounds were treated for 24 h. After addition of an MTT solution  and incubation for 4 h, the supernatants were removed and the formazan crystals were solubilized in 100 μL DMSO. Optical density was determined at 540 nm.Cell viability was assessed by a MTT assay. 3T3-L1 cells were seeded at a density of 1 × 10Male four-week-old C57BL/6 mice weighing 16–21 g were purchased from Central Lab Animal Inc. . The animals were maintained at a constant room temperature of 22 ± 2°C with a humidity of 50 ± 5% and with free access to water and food under a 12 : 12 h light : dark cycle (lights on at 8:00 am). The animals were acclimatized for 4 days before beginning the experiments. All the experimental procedures were conducted in accordance with the relevant guidelines for the care of experimental animals and were approved by the Jeonnam Institute of Natural Resources Research .n = 5 per group): normal diet group , high fat diet group , HFD + uRCB 10 mg/kg/day group , and HFD + uRCB 50 mg/kg/day group . Oral administration of uRCB (10 and 50 mg/kg/day) was continued for 8 weeks from the day of feeding with the HFD. The normal diets consisted of 10% kcal from fat . The HFD consisted of 60% kcal from fat . Body weights and food intakes were measured twice per week.The mice were randomly assigned to four treatment groups consisting of 5 mice each , triglyceride (TG), high-density lipoprotein- (HDL-) cholesterol, low-density lipoprotein- (LDL-) cholesterol, and glucose were measured using an Alere cholesterol LDX system . Serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), and creatinine levels were measured using the appropriate kits . After the blood collection, the adipose tissue  and liver were removed from the mice and immediately weighed.μm particle size, Merck, Darmstadt, Germany) and eluted with a gradient of CHCl3/MeOH  to yield six subfractions (Fr.1–Fr.6) based on the TLC profile (μm). The mobile phase condition consisted of solvent (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile with a linear gradient elution as follows: starting from 97% solvent A, 0–10 min; 50% A, 10–30 min; 10% A, 30–45 min; and 97% A, 45.1–50 min. The sample wavelength was set at 254 nm. A sample injection volume of 5 μL in methanol and a constant flow rate of 0.3 mL/min were used for the analysis of the sample. The mass spectra were acquired with an ESI source . Nitrogen was used as the sheath, and auxiliary gas and helium were used as the collision gas. The ESI MS spectra were acquired in positive ion mode, and a spray voltage of 3.5 kV was employed. The temperature of the heated transfer capillary was 325°C. The mass spectrometer was scanned from m/z 100 to 1,000 in full scan mode, with a gas flow of 11 L/min, nebulizer 35 psi. Six bioactive compounds were confirmed by a HPLC system  with a Capcellpak-C18 column .The BuOH fraction (2.5 g), which showed a strong lipid accumulation inhibitory effect on 3T3-L1 adipocytes, was chromatographed over a silica gel open column  primer and diethyl pyrocarbonate-treated water in a final volume of 20 μL and incubated at 45°C for 60 min. The reaction was stopped by heat inactivation at 95°C for 5 min. Subsequently, the cDNA was amplified with gene-specific primers using a PCR PreMix  according to the manufacturer's instructions. The specific primers that we used in this study are shown in Total RNA was extracted from the 3T3-L1 adipocytes or epididymal adipose tissue using an easy-BLUE total RNA extraction kit  according to the manufacturer's instructions. To synthesize cDNA, 1 n = 6, from three independent experiments with replication. Data were statistically evaluated using Student's t-test or one-way analysis of variance (ANOVA) on GraphPad Prism  software programs. Differences between groups were assessed using Duncan's multiple range tests. Statistical significance was considered at P < 0.05.Data are presented as the mean ± standard deviation (SD), 3, EtOAc, BuOH, and water (μg/mL) were approximately 42.49 ± 3.94% (∗∗P < 0.01), 53.09 ± 0.25% (∗∗∗P < 0.001), and 6.79 ± 1.08% (∗P < 0.05), respectively. Moreover, at low concentrations (10 μg/mL), only the BuOH fraction  significantly inhibited the lipid accumulation during adipocyte differentiation. However, there were no significant differences in the lipid level between the concentrations of the hexane and CHCl3 fractions. The uRCB at concentrations of 0.1, 0.3, 1, 3, 10, 30, and 100 μg/mL inhibited the lipid accumulation rates in a dose-dependent manner to 0.96 ± 2.83%, 2.43 ± 4.12%, 8.14 ± 2.0%, 9.52 ± 4.08%, 21.05 ± 2.63%, 37.11 ± 2.30%, and 56.28 ± 1.10%, respectively (IC50 = 23.88 ± 2.13 μg/mL) (P > 0.05), according to the MTT assay  . These aTT assay , and cau∗∗P < 0.01) and uRCB 50  mice were significantly lower than that of the HFD mice (40.0 ± 0.6 g). The food intake was not significantly different between the HFD group and the uRCB group. The epididymal fat and perirenal adipose tissue weights in the uRCB 10 (1.67 ± 0.09 g and 0.67 ± 0.03 g) and uRCB 50 (1.48 ± 0.04 g and 0.67 ± 0.01 g) mice were significantly decreased compared with those in the HFD group  . The observed decrease in body weight by uRCB treatment without any significant difference in food intake suggests that uRCB has a physiological effect on the processing of food expenditure. We investigated the effects of uRCB on the serum levels of TC, TG, HDL-cholesterol, and LDL-cholesterol in the HFD-induced obese mouse. uRCB (50 mg/kg) effectively decreased the serum levels of TC, TG, LDL-cholesterol, and glucose compared with the HFD group (∗P < 0.05 or ∗∗P < 0.01). In contrast, uRCB significantly increased the levels of HDL-cholesterol in the serum (∗P < 0.05). Moreover, TC and TG in the serum of groups fed diets containing uRCB (10 mg/kg) were significantly lower compared to those in the HFD group (∗P < 0.05). Furthermore, uRCB alleviated liver damage by lowering the levels of two common markers for liver damage, GOT, and GPT, which were, in turn, increased by the HFD treatment. The GOT/GPT activities were not increased in the uRCB-administered groups. Instead, they were decreased. However, the relative weights of the livers were not significantly different between the groups. Finally, to evaluate the potential toxic effects of ingesting uRCB, serum toxicological markers indicating kidney injury were measured at the end of the experimental period. The levels of BUN and creatinine were not significantly changed in uRCB-treated mice compared to the levels in the HFD group. These results suggest that the organ and body weight reductions were not caused by liver and kidney toxicity, further suggesting a beneficial role for Rubus coreanus extracts against obesity.We investigated the antiobesity effect of uRCB  in the HFD-induced obese mouse model for 8 weeks. The body weight, food intake, adipose tissue weights, and serum biochemical parameters of each group are shown in γ is the most important gene during adipogenesis. It plays an important role in adipocyte differentiation, lipid storage, and glucose homeostasis [γ gene expression levels in the epididymal adipose tissue were investigated  and uRCB 50 mg/kg (∗∗P < 0.01) treatment group were significantly decreased compared with those of the HFD group  were 4.56 ± 1.89% (not significant), 3.17 ± 1.72% (not significant), 4.39 ± 1.81% (not significant), 9.08 ± 1.04% (∗P < 0.05), 6.78 ± 0.70% (∗P < 0.05), and 23.29 ± 1.52% (∗∗P < 0.01), respectively. Fr.6 showed a strong lipid accumulation inhibitory effect on the 3T3-L1 adipocytes in a dose-dependent manner (P > 0.05), according to the MTT assay (uRCB was subjected to column chromatography to yield six subfractions (Fr.1–Fr.6). To screen for the optimal inhibition of lipid accumulation in the subfractions of uRCB during adipocyte differentiation, 3T3-L1 preadipocytes were treated with various concentrations of the subfractions of uRCB for 8 days. After incubation, the cells were stained with Oil Red O. The results are shown in t manner . The imat manner . These aTT assay . These rFive compounds were identified by LC-ESI-MS/MS analysis, and each compound was confirmed by comparing their retention times and UV patterns with reference standard compounds using HPLC analysis. The chromatograms of Fr.6 matched those of erycibelline, 5-hydroxy-2-pyridinemethanol, m-hydroxyphenylglycine, ellagic acid, and 4-hydroxycoumarin .μM was not decreased (P > 0.05). However, p-aminophenol led to a 25.86 ± 0.003% and 21.56 ± 0.002% viability compared to the control at concentrations of 30 and 100 μM, respectively (data not shown). Therefore, it was decided that the noncytotoxic concentrations of up to 100 μM of the five bioactive compounds  would be used in the following experiments.To examine the suppressive effects of the six candidates  on 3T3-L1 adipocyte differentiation, we measured the cytotoxicity of various concentrations of the compounds using the MTT assay . The resμM of the five compounds, the lipid accumulation decreased by over 50% compared to the control alone. As shown in μM. The IC50 values of ellagic acid, m-hydroxyphenylglycine, 5-hydroxy-2-pyridinemethanol, 4-hydroxycoumarin, and erycibelline were 52.96 ± 0.60 μM, 89.35 ± 0.88 μM, 93.57 ± 0.94 μM, 95.28 ± 1.57 μM, and 97.25 ± 1.65 μM, respectively.To investigate the inhibitory effects of five bioactive compounds  on adipocyte differentiation, 3T3-L1 cells were treated with various concentrations of the five bioactive compounds for 8 days. After incubation, the lipid accumulation was quantified by Oil Red O staining. The results are shown in γ, C/EBPα, and SREBP-1c. We further studied if the five bioactive compounds regulated the expression of the lipogenic enzymes FAS and ACC. A comparison to fully differentiated adipocytes revealed that the treatment with the five bioactive compounds inhibited the expression of FAS mRNA and induced the downregulation of ACC mRNA expression.To gain a better understanding of the molecular mechanisms underlying the observed antiadipogenic effects, we conducted RT-PCR analysis to examine the influence of the five bioactive compounds on the expression of key transcription factors and lipogenic enzymes . Compare R. coreanus and its possible mechanism based on its maturation. R. coreanus has been used as a medicine or food depending on its ripeness. uRC has been used as a traditional remedy for some chronic diseases, and ripe R. coreanus has been eaten as a fruit.Obesity is a major public health problem arising from a chronic positive energy balance from increased food intake, reduced activity, or a combination of both , 25. ObeAdipose tissue is the primary storage organ for excess energy and is also an endocrine organ. Thus, it is important for the regulation of energy homeostasis , 34. An  R. coreanus [ Prunus mume were decreased compared to those from unripe Prunus mume [ R. coreanus; the citric acid and ellagic acid showed the highest concentration in the uRC [ in vitro and in vivo [γ mRNA expression compared with the mRNA levels in the HFD group.In a previous study, we demonstrated antifatigue effects and antioxidant effects of ripecoreanus . Recentlcoreanus . These rnus mume . Citric  the uRC . Moreove in vivo . Howeverγ and C/EBPα mRNA and TG synthetic enzymes [γ, C/EBPα, and SREBP-1c. In the present study we showed that the five active compound  treatments significantly decreased PPARγ, C/EBPα, and SREBP-1c mRNA expression compared with the differentiated control cells. Recently, some studies reported that 4-hydroxycoumarin has various biological effects, such as anti-inflammatory [μM and 100 μM (data not shown). These results suggest that the five active compounds could play a vital role in the suppression of adipogenesis by downregulating PPARγ, C/EBPα, and SREBP-1c. Adipogenesis is the process of cell differentiation by which preadipocytes become adipocytes. PPARγ, a member of the PPAR subfamily of nuclear hormone receptors, and C/EBPα, a founding member of a large family of leucine zipper transcription factors, are important regulators of adipogenesis, while SREBP-1c regulates the expression of genes such as FAS and ACC that are involved in lipogenesis [γ, C/EBPα, and SREBP-1c and the target genes FAS and ACC. Although the five active compounds are the components that have antiobesity properties in R. coreanus, further studies will be required in order to define the functional compounds that regulate the antiobesity effects of uRC.Woo et al. reported that ellagic acid suppressed 3T3-L1 adipocyte differentiation through a downreduction of the expression of PPAR enzymes . In the ammatory , antitumammatory , anticoaammatory , and antammatory . Jung etammatory . Howeverogenesis . FAS plaγ, C/EBPα, and SREBP-1c, the adipogenesis related gene (ACC), and enzymes (FAS). In addition, uRCB decreased the body weight, adipose tissue weight , and serum TC/TG, glucose, and LDL-cholesterol levels in HFD-induced obese mice. Taken together, uRC, which has been used as a traditional remedy, might be used as a potential functional material for the prevention and treatment of obesity because of its antiadipogenic effects. An animal study is under way to evaluate the antiobesity effects of active compounds using a HFD-induced obese mouse model.In conclusion, this study is the first to report that uRCB and its five active compounds  inhibit adipocyte differentiation through the suppression of transcriptional factors, including PPAR


We fused enhanced green-fluorescent protein to α-tubulin (ZtTub2), to ZtPeb1, a homologue of the mammalian plus-end binding protein EB1, and to ZtGrc1, a component of the minus-end located γ-tubulin ring complex, involved in the nucleation of microtubules. In vivo observation confirms the localization and dynamic behaviour of all three markers. These marker proteins are useful tools for understanding the organization and importance of the microtubule cytoskeleton in Z. tritici.The microtubule cytoskeleton supports vital processes in fungal cells, including hyphal growth and mitosis. Consequently, it is a target for fungicides, such as benomyl. The use of fluorescent fusion proteins to illuminate microtubules and microtubule-associated proteins has led to a break-through in our understanding of their dynamics and function in fungal cells. Here, we introduce fluorescent markers to visualize microtubules and accessory proteins in the wheat pathogen  The fully sequenced Z. tritici wild-type isolate IPO323 (n EHA105  was usede IPO323  was used2.2Z. tritici strain IPO323 (http://genome.jgi.doe.gov/Mycgr3/Mycgr3.home.html) using protein BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the U. maydis proteins sequences of Tub1 (accession number: XP_757368.1), Peb1 (http://www.ncbi.nlm.nih.gov/pubmed) and comparisons were done using CLUSTAL W  and EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). This revealed that a mistake in the annotation of ZtGrc1, which is predicted without a stop codon. We choose the next down-stream stop codon as the end of the open reading frame. This extended the predicted protein length by 10 amino acids. This corrected ZtGrc1 sequence was used for further analyzis and cloning. Domain structures were analyzed in PFAM (http://pfam.xfam.org/search/sequence). Finally, phylogenetic trees were generated in MEGA5.2, using Neighbor-Joining algorithm, followed by 1000 bootstrap cycles , 706 bp of exon 3 of sdi1 gene , 1149 bp Z. tritici tub2 promoter  and 1510 bp hygromycin resistance cassette , 3′ end of 686 bp eb1  of H267L mutated, carboxin-resistance conferring allele of sdi1 gene  and a 1510 bp hygromycin resistant cassette , 3′ end of 1049 bp grc1  of H267L mutated allele of sdi1 gene  and a 1510 bp hygromycin resistant cassette , PvuII (for pHEB1eGFP transformants) and XmnI (for pHGRC1eGFP transformants) and separated on a 1.0% agarose gel and capillary transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech). 1014 bp sdi1 probe (3′ end of the Rsdi1 gene and sdi1 terminator) was generated by with primers SK-Sep-10 and SK-Sep-13; eb1 probe (downstream non coding sequence of the eb1 gene) was generated with primers SK-Sep-100 and SK-Sep-51; grc1 probe (3′ end of the grc1 gene) was generated with primers SK-Sep-37 and SK-Sep-38; The vectors pCeGFPTub2, pCmCherryTub2, pHEB1eGFP and pGrc1eGFPwere transformed into k method  and A. tocedures . 3 μg of2.8z resolution of 0.2 μm and an exposure time of 150 ms. The 488 nm laser was used at 100%. The final images are maximum projections generated in MetaMorph . The plus-end marker EB1 was imaged in time laps movies with an exposure time of 250 ms and a time interval of 500 ms. The 488 nm laser was used at 75%. To co visualize the microtubules in red and the plus end marker in green, z-stacks with a z resolution of 0.2 μm and an exposure time of 250 ms were taken. The 488 nm laser was used at 75% and the 561 nm laser was used at 100% output power. The image were 2D deconvolved followed be generating a maximum projection using MetaMorph . The microtubules were depolymerised in the strains IPO323_CeGFPTub2 and IPO323_HEB1eGFP by incubating the cells in YG media containing 300 μM benomyl  for 45 min at 18 °C with 200 rpm. Treated cells were placed onto a 2% agar cushion containing 300 μM benomyl  and directly imaged using 150 ms exposure time and the 488 nm laser at 100%. To visualize the Grc1 at the spindle pole bodies Grc1-eGFP was co-localized with mitotic spindles labelled with mCherry-Tub2 by generating z-stacks with a z resolution of 0.2 μm and an exposure time of 150 ms. The 488 nm laser was used at 40% and the 561 nm laser was used at 75% output power. The final images are maximum projections generated in MetaMorph .Fluorescence microscopy was performed as described in 33.1Z. tritici homologues of the U. maydis α-Tubulin, Tub1 (U. maydis sequences against the Z. tritici published genome sequence of IPO323 at the Joint Genome Institute (http://genome.jgi.doe.gov/Mycgr3/Mycgr3.home.html). This revealed the putative α-tubulin , which is 99.8% identical to the described α-tubulin from a different strain of Z. tritici . In addition, ZtTub2 and ZtEB1 share domains with their counterparts in U. maydis . In contrast, the putative γ-tubulin ring complex protein ZtGrc1 shared only 17.6% amino acid sequence identity with UmGrc1, 18.8% with Spc97 from S. cerevisiae (NCBI accession number: EGA58404.1) and 17% with Spc98 from S. cerevisiae (NCBI accession number: EEU08751.1). However, ZtGrc1 contains a Spc97/Spc98 family domain (P = 3.5e−133). Therefore, we felt confident in establishing these Z. tritici as markers for microtubules and their plus- and minus end.In this study we set out to establish fluorescent marker proteins for microtubules and their plus- and minus-ends. We chose α-tubulin, an EB1-like plus-end binding protein and a γ-tubulin ring complex protein A. We idein, Tub1 , the EB1in, Tub1  and the in, Tub1  by perfo as Tub1 , we name as Tub1 B. The pr3.2Z. tritici, we constructed firstly the vector pCeGFPTub2 that allows the expression of eGFP-Tub2 fusion protein under the control of the constitutive Z. tritici α-tubulin (tub2) promoter . It allows A. tumefaciens-based transformation into Z. tritici, which is based on the 25 bp imperfect directional repeat sequences of the T-DNA borders . Incorporation by homologous recombination mutates the sdi1 gene and integrates the eGFP-Tub2 construct into the sdi1 locus  to the genomic loci of both genes, which ensures native expression levels of the fluorescent proteins ZtEB1-eGFP and ZtGrc1-eGFP. During homologous recombination in Z. tritici, the partial 3-prime sequences of the open-reading frame of eb1 and grc1 serve as left flanks, whereas 3′ non-coding regions of eb1 and grc1 serve as right flanks  promoter . This velocity corresponds well with the microtubule polymerization velocities in other fungi , nptII  or bar (phosphinothricin acetyltransferase; Basta-resistant; see Finally, we constructed a set of vectors that carried a second resistance cassette. This was done to provide the opportunity to combine any of the vectors with other constructs or fluorescently labelled components. Firstly, we generated vector pHeGFPTub2, designed for random ectopic integration of the eGFP-Tub2 fusion construct E. This vcassette F and shacassette B. The ve4in vivo dual-color imaging. These markers will be useful tools to understand the organization and dynamic behaviour of the microtubule cytoskeleton in Z. tritici. This knowledge, in combination with the various vectors we provide, will help to characterize mutant phenotypes in Z. tritici. In addition, our tools will inform mode of action studies on new anti-fungals, aiming to develop novel control strategies for Z. tritici blotch on wheat.The microtubule cytoskeleton performs essential roles in mitosis and interphase of fungal cells. It is, therefore, a target for fungicide development . Here, w


Papaver somniferum, and genes encoding enzymes responsible for the synthesis of noscapine have been recently discovered to be clustered on the genome of P. somniferum. Here, we reconstitute the noscapine gene cluster in Saccharomyces cerevisiae to achieve the microbial production of noscapine and related pathway intermediates, complementing and extending previous in planta and in vitro investigations. Our work provides structural validation of the secoberberine intermediates and the description of the narcotoline-4′-O-methyltransferase, suggesting this activity is catalysed by a unique heterodimer. We also reconstitute a 14-step biosynthetic pathway of noscapine from the simple alkaloid norlaudanosoline by engineering a yeast strain expressing 16 heterologous plant enzymes, achieving reconstitution of a complex plant pathway in a microbial host. Other engineered yeasts produce previously inaccessible pathway intermediates and a novel derivative, thereby advancing protoberberine and noscapine related drug discovery.Noscapine is a potential anticancer drug isolated from the opium poppy  Noscapine is a potential anticancer drug that is traditionally isolated from the opium poppy Papaver somniferum. Here, Li and Smolke reconstitute the noscapine gene cluster in Saccharomyces cerevisiae, to achieve the microbial production of noscapine and related pathway intermediates, and provide new insights into the biosynthesis of noscapine. Plant natural products span a large and diverse class of compounds with critical ecological functions25678910in planta gene silencing studies are used to verify gene identity and identify enzyme function within the pathway; and in vitro biochemical studies are used to understand the function and study enzymatic mechanisms12In planta characterization of individual enzyme activities can be impacted by the complex endogenous protein and metabolite background, and in vitro characterization can be limited by the availability of pathway intermediates as substrates and standards, many of which are difficult to synthesize chemically or extract from producing plants. A number of tools have recently been developed that enable the efficient reconstitution of known plant natural product pathways in the budding yeast Saccharomyces cerevisiae14151618197212214151617in planta and the downstream in vitro characterization of enzyme mechanism.Currently, L-tyrosine through scoulerine3739404142434445Noscapine is a potential anticancer medicine with a long-established safety record as an oral drug26272831323334313233341 was recently discovered in Papaver somniferumP. somniferumin vitro characterization experiments were able to correct the ordering of several steps  and proposed an updated biosynthetic pathway474849A 10-gene cluster involved in the biosynthesis of noscapine, S. cerevisiae. Our synthetic approach provides experimental support for the previously proposed reaction matrix of the final three steps of the pathway and suggests that the remaining 4′-O-methylation step is catalysed by two different O-methyltransferases together, and to the best of our knowledge, is likely to be the first known O-methylation natively catalysed by an O-methyltransferase heterodimer. Our reconstituted noscapine gene cluster also provides sufficient levels of pathway intermediates to support structural verification, which allows us to validate the secoberberine structures synthesized by CYP82X1. In addition, the 14-step biosynthesis of noscapine from the early benzylisoquinoline alkaloid norlaudanosoline is achieved in yeast by optimizing the expression of 16 plant enzymes, reaching a titre of 1.64±0.38 μM noscapine.To provide an alternative route to the production of noscapine, and to overcome the inaccessibility of many pathway intermediates and potentially interesting derivatives, we reconstitute the noscapine gene cluster in the yeast host S)-scoulerine to noscapine. The cluster encodes four cytochrome P450 monooxygenases , three methyltransferases , one carboxylesterase (PsCXE1), one short-chain dehydrogenase/reductase (PsSDR1), and one acetyl transferase (PsAT1), making it one of the most complex plant natural product pathways described to dateP. somniferumin vitro biochemical characterization studies50S)-scoulerine to (S)-canadine, 2, which is then converted to N-methylcanadine, 3, by tetrahydroprotoberberine cis-N-methyltransferase from P. somniferum (PsTNMT), and this compound is subsequently hydroxylated to 1-hydroxy-N-methylcanadine, 4, by 1-hydroxy-N-methylcanadine synthase from P. somniferum (CYP82Y1).The noscapine gene cluster encodes 10 enzymes to convert  supplemented with 250 μM of racemic 2, which is assumed to contain equal amounts of each enantiomer. The medium is then directly analysed for pathway metabolites after 72 h of growth by liquid chromatography/mass spectrometry (LC/MS) analysis. Previous studies indicated that using galactose as a carbon source improves alkaloid production in yeast from fed substrates, which may be related to the metabolic or transcriptional responses induced by galactosein vivo assays in this work.We started the reconstitution of the noscapine biosynthetic pathway from canadine, as the biosynthesis of Arabidopsis thaliana (AtATR1) that was previously shown to function with diverse plant P450s  when that enzyme is expressed from the GAL1 promoter at 30 °C -cis-N-methylstylopine-1-hydroxylase (MSH), resulted in slightly enhanced activity from the chimeric protein (26.9±2.0 μM) when expressed downstream of the GAL1 promoter at 30 °C (4 at lower temperature (P=0.0021) and the N-terminus engineering (P=0.0436) are both significant through a standard two-way analysis of variance  from 50s refs , 55. Whe2Y1 refs , 56. Nuced yeast . However °C ref. . To ensuat 30 °C . Both CYvariance . The higin planta studies performed in P. somniferum indicated that inactivating CYP82X2 leads to the synthesis of 4′-O-desmethylmacrantaldehyde5′. The subsequent in vitro study indicated that CYP82X1 is responsible for the conversion of 4 to 5′ , which is confirmed by NMR analyses (PGK1) at 25 °C .We first engineered the analyses ; howeveranalyses . We optianalyses  when theat 25 °C , compare4-producing yeast strain, 5 (m/z+=386)  when the enzyme was expressed from the HXT7 promoter at 25 °C   was observed when the enzyme was expressed from relatively weak promoters (ADH1 and CYC1) and late promoter (HXT7) at 25 °C  of 2.8 min that agrees with 4′-O-desmethlpapveroxine, 6′, or narcotolinehemiacetal, 8, in the 7-producing strain   is synthanalyses . We optiat 25 °C . By engianalyses . The exp/z+=444) . Subsequveroxine . To ensuat 25 °C . When weg strain . This pe CYP82X1 . This daN-methylcanadine implies that the conversion from 1,8-dihydroxy-13-O-acetyl-N-methylcanadine to 7 is either highly efficient in yeast, or more likely, that this reaction is facilitated by CYP82X1  resulted in accumulation of 4′-O-methylated 3-O-acetylpapaveroxine, 7′, and narcotinehemiacetal, 8′, respectively. However, an in vitro study of PsMT2 indicated that PsMT2 has no activity towards narcotoline, but functions as a S9OMT with strict substrate specificityin vitro biochemical study supports the activity of PsCXE1 in the conversion of 7′ to 8′ and PsSDR1 in the conversion of the m/z+=402 peak synthesized spontaneously from 7 to narcotoline, 9 (ref. S)-scoulerine-9-O-methylation), the identity of the 4′-O-methyltransferase required for the 4′-O-methylation and the sequence of the enzymatic steps catalysed by the 4′-O-methyltransferase, PsCXE1 and PsSDR1 remain in question.According to previous , 9 ref. . Thus, t7-producing strain to express PsMT2. However, when grown in the presence of 2 at 25 °C, the engineered strain did not synthesize 7′ or any methylated intermediates (7-producing strain to express PsCXE1 downstream of a strong promoter (PYK1 and GPD) and observed synthesis of 8  column, we were unable to purify 8 and conduct NMR analyses to further confirm the structure. The titre of 8 in the medium was therefore estimated by comparison to the standard curve of structurally similar 9.We first engineered the mediates . Then, w7-producing strain to express PsCXE1 and PsSDR1, and observed the efficient synthesis of 9 from 8 . Previous in vitro characterization studies demonstrated that PsMT3 showed no activity on narcotoline and proposed that this enzyme acts as a S9OMT, similar to PsMT1  and 8′  in these strains  by increasing the expression of CYP82X2, as suggested in the aforementioned optimization for the synthesis of 5 (The extracellular titre of noscapine reaches 2.2±0.3 μM when the yeast strain is engineered to express each enzyme on an optimized expression construct and grown in the presence of 250 μM racemic 2 . The extsis of 5 .S)- (98% purity) or (R)-(96% purity) canadine to our engineered yeast strains that synthesize the major pathway intermediates to roughly estimate the promiscuity of the pathway enzymes on each enantiomer. Interestingly, although (S)-canadine is the natural substrate of the pathway in opium poppy, (R)-canadine can also be converted through the pathway to noscapine when fed independently, but at much lower efficiencies than (S)-canadine in the early steps of the pathway (4 is synthesized when fed with (S)-canadine and (R)-canadine, respectively). However, we also observed that the ratio between (S)-enantiomer and (R)-enantiomer decreases along the pathway (R)-canadine.We then fed 125 μM  intermediate, 12, has recently been reported in S. cerevisiae2 to noscapine enables the connection of these two pathway parts to achieve the microbial biosynthesis of noscapine from 12. Since Ps6OMT can function together with PsMT2 as the narcotoline-4′-O-methyltransferase, yeast strains were engineered to express AtATR1, Ps6OMT, 3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase from P. somniferum (Ps4′OMT), coclaurine N-methyltransferase from P. somniferum (PsCNMT), berberine bridge enzyme from P. somniferum (PsBBE), S9OMT from P. somniferum (PsS9OMT or PsMT1), CAS from Coptis japonica (CjCAS), PsTNMT, CYP82Y1, CYP82X2, PsAT1, CYP82X1, PsCXE1, PsSDR1 and PsMT2 without or with PsMT3  and phthalideisoquinoline (noscapine and 9) alkaloids from fed 2 or 12. The activities of enzymes encoded in the gene cluster reconstituted in yeast agree well with the in planta and in vitro biochemical characterization studies, and provides additional insights into the biosynthesis of noscapine. Our work also enables the supply of many pathway intermediates and derivatives for structural confirmation and further investigation.The 10-gene cluster encoding enzymes responsible for the conversion of scoulerine to noscapine was first discovered and verified in 2, to our strains, which is assumed to be composed of equal amounts of each enantiomer. The substrate specificity of the noscapine pathway enzymes towards different enantiomers has not previously been investigated4748in vitro. In addition, individual enantiomer standards of pathway intermediates between canadine and noscapine are unavailable. Thus, it is difficult to estimate the ratio between each enantiomer along the noscapine pathway. We fed (S)- and (R)-canadine to our engineered yeast strains, and the data indicated that although our racemic canadine-fed strains mainly produce (S)-enantiomers of the pathway metabolites, (R)-enantiomers may also be present in small quantities. However, considering that the (R)-canadine is of 96% purity, it is difficult to estimate the exact percentage of pure (R)-enantiomers in strains fed with (R)-canadine. In addition, when fed with racemic 2, the conversion of (S)- and (R)-canadine to the pathway intermediates may shift from when they are fed separately to the engineered yeast strains, and whether or not (R)-canadine can be converted when both enantiomers are present is unknown.We primarily fed racemic canadine, de novo production of reticuline in yeast64de novo production of noscapine and related molecules could provide a cost-efficient process for the production of these valuable molecules. However, no less than eleven enzymes, including four cytochrome P450s, will need to be introduced into the reticuline-producing strain for the de novo synthesis of noscapine. The full de novo pathway to noscapine is thus composed of up to 18 heterologous enzymatic steps from tyrosine and at least 23 different heterologous enzymes, including 5 cytochrome P450s. Our data indicates that as the heterologous pathway introduced into yeast includes more P450s, the conversion efficiency of each P450 will decrease, which may be due to challenges associated with endoplasmic reticulum loading and cofactor regeneration. Therefore, introducing a pathway comprising the expression of at least 5 cytochrome P450s will require further engineering of the yeast background to increase efficiencies. In addition, the reported strains produce relatively low levels of reticuline from tyrosine with titres reported at <100 μg l−1 .Throughout our study, we measured the pathway metabolite levels in the growth medium from the engineered yeast strains. Our data indicates that most metabolites were transported out of the yeast cells efficiently . Howeverin planta and in vitro studies implied that the synthesis of 7 to noscapine is likely to be branched into several routesin planta study found that the enzymes catalysing the final three steps of morphine biosynthesis are among the most abundant active proteins in the latex sub-proteome, which indicates that the final three steps are likely to principally occur in the laticifersin vitro and in planta in which the pathway enzymes function can be different, and these differences can result in distinct substrate preferences, enzyme activities and product transportation processes. Thus, observations of pathway sequence and efficiency across these different contexts should ultimately be considered with regard to these differences.We observed that PsSDR1 or PsMT2/PsMT3 could catalyse the final step in the synthesis of noscapine in yeast . Previouin planta investigations are essential for validating that PsMT2 and PsMT3 form a heterodimer in opium poppy, and studying the native functions of PsMT2, PsMT3, Ps6OMT and their dimers.Our study also uncovered a unique O-methylation step that is likely to be natively catalysed by an O-methyltransferase heterodimer in opium poppy. While previous studies demonstrated that plant O-methyltransferase heterodimers can exhibit different substrate specificity from the corresponding homodimers in synthetic contexts59in planta and in vitro characterizations of plant natural product pathways to provide a complementary approach for better understanding and engineering the biosynthetic potential of plants. The yeast-based plant pathway reconstitution also has the advantage of simultaneously building production strains for potentially valuable compounds, including all intermediates along a pathway and novel derivatives thereof. While the end-product noscapine accumulates in plants at low levels, most of the pathway intermediates, which can serve as interesting lead compounds for drug discovery, are in exceedingly low abundance in the producing plants, and thus not accessible. In addition, enzymatic steps can be readily removed or introduced in the yeast platform to enable the synthesis of novel structures such as 10. Our work demonstrates the potential of synthetic biology as a powerful tool for aiding complex pathway elucidation, while enabling microbial production of valuable natural or unnatural medicinal compounds originated from plants.Step-wise pathway reconstitution in yeast can be applied to bridge 2, norlaudanosoline (98% purity), 12, and reticuline were purchased from Finetech Industry Limited, Santa Cruz Biotechnology and Specs, respectively. (R)- and (S)-enantiomers of 2 were purchased from Toronto Research Chemicals. S-Adenosyl methionine (80% purity) and noscapine (97% purity), 1, were purchased from Sigma-Aldrich. Narcotoline (>95% purity), 9, was purified from 9-producing yeast strain in this study as described below. For DNA manipulation and amplification, we used chemically competent or electrocompetent E. coli strains TOP10 and ccdB Survival 2 T1R purchased from Life Technologies. E. coli strains were grown at 37 °C in lysogeny broth medium obtained from Fisher Scientific supplemented with 100 mg ml−1 ampicillin  or 50 mg ml−1 kanamycin (Fisher Scientific) for plasmid maintenance. All engineered yeast strains described in this work, as listed in leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) −1 G418 sulfate  or 200 mg l−1 Hygromycin B (Life Technologies) were used in YPD medium for selection. BL21(DE3) and Ni-NTA agorose resin used for E. coli protein expression and hexa-histidine tagged protein purification were kindly provided by Professor Chaitan Khosla, and T7 Tag Affinity Purification Kit used for T7-tagged protein purification was purchased from EMD Millipore. NuPAGE Novex 4–12% Bis-Tris Protein Gels  used for purified protein SDS–PAGE analysis and western blot were purchased from Life Technologies and Color Prestained Protein Standard  used as the protein marker was obtained from New England Biolabs. The Anti-6X His tag antibody (horseradish peroxidase (HRP), ab1269) and Anti-T7 tag antibody  used for western blot of PsMT2/PsMT3 and PsMT2/Ps6OMT heterodimers were purchased from Abcam PLC. Size-exclusion chromatography was performed on a BioSep-SEC-s4000 column , and the column was equilibrated with Gel Filtration Markers Kit for Protein Molecular Weights 12,000–200,000 Da purchased from Sigma.Racemic canadine (98% purity), S. cerevisiae Advanced Gateway Destination Vector Kit (Addgene)Plasmid DNA was prepared using the QIAprep Spin Miniprep Kit (QIAGEN) and Econospin columns (Epoch Life Science) according to manufacturer's protocols. PCR reactions were performed with Taq polymerase, PfuUltra Hotstart DNA Polymerase (Agilent Technologies) and Expand High Fidelity PCR System (Roche Life Science) according to manuacturer's protocols. PCR products were purified by Zymoclean Gel DNA Recovery Kit (Zymo Research). All DNA constructs were confirmed through DNA sequencing by Elim Biopharmaceuticals Inc. Restriction enzymes (NEB) and T4 ligase (NEB) were used to digest and ligate the DNA fragments, respectively. BP Clonase II Enzyme Mix, Gateway pDONR221 Vector and LR Clonase II Enzyme Mix (Life Technologies) and the CYP82Y1, CYP82X2, PsAT1, CYP82X1, PsCXE1, PsSDR1, PsMT2 and PsMT3) were codon-optimized for expression in S. cerevisiae using the GeneArt GeneOptimizer programme (Life Technologies) and synthesized by GeneArt (Life Technologies) or integrated DNA technologies (IDT). The synthesized genes and Ps6OMT and Tf6OMT (amplified from previous constructsS. cerevisiae Advanced Gateway Destination Vector KitPsAT1 was amplified with primer PsAT1-F (5′- caccagaacttagtttcgacgg attctagaactagttatacaatggctaccatgtcatctgctgc -3′) and PsAT1-R (5′- attacatgactcgaggtcgacggtatcgat aagcttttagaataattgcaagatttctctcaagtggtgttc -3′) and inserted into pAG415GPD-ccdB digested with SacI and HindIII with yeast in vivo homologous recombination to yield pCS3124.Genes encoding CYP82Y1, CYP82X2, PsAT1, CYP82X1, PsCXE1, PsSDR1, PsMT2 and PsMT3 (HXT7 promoter was cloned from pCS2658 with primers HXT7p-SacI-F (5′- aaaaaagagctcctcgtaggaacaatttcgggccc -3′) and HXT7p-SpeI-R (5′- aaaaaaactagtgttttttgattaaaattaaaaaaacttttt gtttttgtgtttattctttgttc -3′). The resulting DNA fragment was digested with SacI and SpeI and inserted in pAG413GPD-ccdB, pAG414GPD-ccdB and pAG416GPD-ccdB (digested with SacI and SpeI) to yield pAG413HXT7-ccdB (pCS3111), pAG414HXT7-ccdB (pCS3112) and pAG416HXT7-ccdB (pCS3113) (ccdB-CamR cassette was amplified from pAG413GPD-ccdB by ccdB-gibson-F (5′- gggaacaaaagctggagctcccgcggcccatcacaagtttgtacaaaaaagctgaac -3′) and ccdB-gibson-R (5′- ctatagggcgaattgggtaccgtttaaacgcggccgccccatcaccactttgtacaagaaagctg -3′). The DNA fragment was inserted into vectors amplified through PCR from pAG413GPD-ccdB, pAG414GPD-ccdB, pAG415GPD-ccdB and pAG416GPD-ccdB by pAG-gibson-F (5′- gagctccagcttttgttccctttag -3′) and pAG-gibson-R (5′- gtacccaattcgccctatagtgagtcg -3′) using Gibson assembly to yield pAG413-ccdB (pCS2811), pAG414-ccdB (pCS2812), pAG415-ccdB (pCS2813) and pAG416-ccdB (pCS2814), respectively (Construction of new destination vectors for Gateway cloning. The pCS3113) . pCS3116pCS3113) . The ccdectively . pCS3141ectively  was recoectively .S. cerevisiae (L14D), a cytochrome P450 reductase partner CPR from Candida maltose (CmCPR) and cheilanthifoline synthase from E. californica (EcCFS) using Gibson assembly to yield pCS2842, and 3121-3123 encoding CYP82Y1A, CYP82Y1B, CYP82Y1C and CYP82Y1D -cis-N-methylstylopine-1-hydroxylase (MSH), lanosterol 14-demethylase from CYP82Y1D , respect180–3182 . Similar180–3182 .CYP82Y1A was inserted into pCS2657–2661 and 2663–2664 , pAG414HXT7-ccdB (pCS3112) and pAG416HXT7-ccdB (pCS3113) to construct expression cassettes regulated by the GPD promoter and HXT7 promoter  and R (5′- atctcagtggtggtggtggtggtgctcgagtgggtaagcaacgataatagatggcatg -3′), and PsMT3-pET-F (5′- tttgtttaactttaagaaggagatatacatatggaagttgtctccaa gatcgatcaag -3′) and R (5′- catttgctgtccaccagtcatgctagccatgtgtggataagcttcaataacggattgcaag -3′), and Ps6OMT-pET-F (5′- tttgtttaactttaagaaggagatatacatatggaaacagtaagcaagattgatcaacaaaacc -3′) and R (5′- catttgctgtccaccagtcatgctagccatataagggtaagcctcaattacagattggacag -3′), respectively, to generate DNA fragments encoding PsMT2 with hexa-histidine tag, PsMT3 with T7 tag and Ps6OMT with T7 tag at the C terminus. The PsMT2 fragment was then inserted into pET28a (amplified by pET28_gibson_F (5′- tatatctccttcttaaagttaaacaaaattatttctagagggg -3′) and pET28 gibson R (5′- tgcttacccactcgagcaccaccaccacc -3′)) using Gibson assembly to construct pCS3306  were PCR amplified and incorporated into selected loci to generate CSY1000 and CSY1054  and GPD-pCS1056-R (5′- tatacgaagttatattaagggttgtcgacgc ggccgcaaattaaagccttcg -3′), and inserted into pCS1056 and PCR amplified by pCS1056-F (5′- gcggccgcgtcgacaacccttaatataacttcgtataatgtatg -3′) and pCS1056-R (5′- gtttaaaccatatggtgcactctcagtacaatctgc -3′) using Gibson assembly to yield pCS3145 (HXT7-CYP82Y1A-TCYC1 (pCS3146), PHXT7-CYP82X2-TCYC1 (pCS3147) and PGPD-PsAT1-TCYC1 (pCS3148) integration cassettes were constructed using a similar procedure  and HIS3 pUG R (5′- agtatcatactgttcgtatacatacttactgacattcataggtatacatatatacacatgtatatatatcgtatgctgcagctttaaataatcggtgtcactacgactcactatagggagacc -3′), Leu2 pUG F (5′- ttttccaataggtggttagcaatcgtcttactttctaacttttcttaccttttacatttcagcaatatatatatatatatttcaaggatataccattctaatgagattgtactgagagtgcacc -3′) and Leu2 pUG R (5′- taccctatgaacatattccattttgtaatttcgtgtcgtttctattatgaatttcatttataaagtttatgtacaaatatcataaaaaaagagaatctttttacgactcactatagggagacc -3′), and Trp1 pUG F (5′- gtatacgtgattaagcacacaaaggcagcttggagtatgtctgttattaatttcacaggtagttctggtccattggtgaaaagattgtactgagagtgcac -3′) and Trp1 pUG R (5′- tatgtttagatcttttatgcttgcttttcaaaaggcctgcaggcaagtgcacaaacaatacttaaataaatactactcagcgactcactatagggagacc -3′).Single-gene chromosomal integration. The AtATR1 integration cassette was PCR amplified from pCS1056 ref.  with URA pCS3145 . PHXT7-Crocedure . PGPD-Psd CSY999 . The prig for 5 min, pellets resuspended in fresh SDM with 2 mM norlaudanosoline and 2% galactose, and incubated at 25 °C for 96 h before metabolite analysisFor all the assays reported in this work, yeast were grown in 500 μl media in 96-well plates  covered with AeraSeal film (Excel Scientific), at 480 r.p.m., 80% humidity in a Kuhner Lab-Therm LX-T plate shaker. For step-wise reconstitution of the noscapine biosynthetic pathway from canadine, yeast strains were first cultured overnight in 500 μl SDM with 2% dextrose. 5–10 μl of the overnight seed culture was inoculated in fresh 500 μl SDM with 250 μM canadine and 2% galactose, and incubated at 25 or 30 °C for 72 h before metabolite analysis of the growth medium. For the synthesis of noscapine from canadine and norlaudanosoline, 5–10 μl of the overnight seed culture was inoculated in 500 μl 5 × concentrated SDM  and incubated at 25 °C for 48 h ref. . The cel3OH  to 60% CH3OH  over 7 min with a flow rate of 0.5 ml min−1. On the Agilent 6420 triple quad LC–MS, metabolites in the medium were separated on a linear gradient of 10% CH3CN  to 50% CH3CN  over 5 min with a flow rate of 0.4 ml min−1 for scan mode; or 10% CH3CN  to 40% CH3CN  over 5 min with a flow rate of 0.4 ml min−1 for multiple reaction monitoring (MRM) mode. The identity of each compound was verified by comparing MS/MS spectra of samples and literature or standards. The conversion of each enzymatic step was estimated by comparing the integrated peak area of extract ion chromatograms (EICs) to a standard curve. The quantities of 3, 4, 5, 5′, 6, 7 and 9 were estimated by comparing to the stand curves of the corresponding purified and NMR verified standards. The quantities of 8, 8′, 10 and 11 were estimated by comparing to the standard curves of structurally similar 9, 1, 2 and 4, respectively, due to the lack of standards. Noscapine levels were quantified by MRM using the highest characteristic precursor ion/product ion transition  and an Agilent 6420 triple quad LC–MS  using positive ionization. On the Agilent 6300 Series Ion Trap LC–MS, metabolites in the medium were separated on a linear gradient of 20% CHansition , and comg for 5 min, pellets resuspended in fresh SDM with 2 mM norlaudanosoline and 2% galactose, and incubated at 25 °C  in a volume equal to that of the cell pellet . The medium and cell pellet extracts were analysed on an Agilent 6420 triple quad LC–MS  using positive ionization. The substrate and pathway metabolites reticuline, scoulerine, tetrahydrocolumbamine, 1–9 and 12 were monitored by MRM using the highest characteristic precursor ion/product ion transition  and incubated at 25 °C for 48 h ref. . The cel °C ref. . Three 1ansition . The qua1, 3, 4, 5, 5′, 6, 7 and 9 for structural elucidation by NMR, and for in vitro characterization of the PsMT2/PsMT3 heterodimer, the 500 μl in vivo assay was scaled up to 200–1,000 ml with each 100 ml of medium containing 2% galactose and 12 mg canadine, and cultured in 500 ml shake flask at 25 °C for 120 h. Yeast culture medium was separated from cell pellets by centrifugation at 4,416g at 5 min. The supernatant was incubated with 20 g of Amberlite XAD4 (Sigma-Aldrich) overnight. XAD4 resins were then collected through vacuum filtration, and metabolites were recovered from the resin by extracting with 50 ml MeOH for three times. The resultant organic extracts were combined and evaporated to dryness, redissolved in 20% CH3CN . 1, 3, 4, 5′, 6 and 7 were purified by reverse-phase HPLC  on an isocratic elution with 20% CH3CN (v/v) over 15 min and 95% CH3CN (v/v) for 5 min in water (0.1% formic acid) at a flow rate of 4 ml min−1; and 5 and 9 were purified by reverse-phase HPLC  on an isocratic elution with 15% CH3CN (v/v) over 15 min and 95% CH3CN (v/v) for 5 min in water (0.1% formic acid) at a flow rate of 4 ml min−1. The 1H, 1H–1H correlation (COSY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) NMR spectra of 6 and 7, the 1H–1H correlation (COSY) NMR spectra of 5′ were performed on the Varian Inova 600 spectrometer using CD3CN as the solvent. The 13C-NMR spectrum of 7 was performed on the Varian ui500 spectrometer using CD3CN as the solvent. The 1H, HMQC and HMBC NMR spectra of 5′ were performed on the 800 MHz Agilent (Varian) VNMRS spectrometer using CD3CN as the solvent. The 1H, HMQC and HMBC NMR spectra of 5 were performed on the Varian Inova 600 spectrometer using CDCl3 as the solvent. The 1H-NMR spectra of 3 and 4 were performed on the Varian Inova 600 spectrometer using CD3CN as the solvent, and the 1H-NMR spectra of 1 and 9 were performed on the Varian Inova 600 spectrometer using DMSO-d6 as the solvent. High-resolution mass spectrometry data were obtained on a coupled Agilent 6520 Accurate-Mass Q-TOF ESI mass spectrometer in positive ion mode, and analysed using Mass Hunter Qualitative Analysis software (Agilent).To obtain sufficient amount of 3 were as follows: 1H-NMR : δ 7.05 , 6.95 , 6.79 , 6.75 , 6.00 , 5.98 , 4.73 , 4.60 , 4.56 , 3.85 , 3.83 , 3.68 , 3.46 , 3.42 , 3.08 , 3.21  and 3.16 . This agrees with previous reported NMR data of 3 (refs m/z): [M]+ calcd. for C21H24Cl2NO4+, 354.1705; found, 354.1706.NMR data of f 3 refs , 71. HRM4 were as follows: 1H-NMR : δ 7.03 , 6.91 , 6.28 , 5.85 , 5.22 , 4.67 , 4.78 , 3.85 , 3.83 , 3.60 , 3.31 , 3.49 , 2.86 , 3.19 , 3.06  and 3.14 . This agrees with previous reported NMR data of 4 (refs m/z): [M]+ calcd. for C21H24Cl2NO5+, 370.1654; found, 370.1655.NMR data of f 4 refs , 71. HRM5′ were as follows: 1H-NMR : δ 10.22 , 7.14 , 6.93 , 6.23 , 5.88 , 3.90 , 3.87 , 3.83 , 3.22 , 3.20 , 2.90 , 2.76 , 2.62 , 2.34 , 2.19 . HRMS (m/z): [M+H]+ calcd. for C21H24NO6+, 386.1604; found, 386.1597.NMR data of 5 were as follows: 1H-NMR : δ 7.20 , 6.95 , 6.05 , 5.86 , 5.28 , 4.93 , 4.88 , 3.874 , 3.865 , 3.38 , 3.34 , 3.15  and 2.92 . This agrees with previous reported NMR data of 5 (ref. m/z): [M]+ calcd. for C21H24NO6+, 386.1604; found, 386.1598.NMR data of f 5 ref. . HRMS : δ 7.08 , 7.03 , 6.21 , 6.13 , 5.84 , 5.48 , 4.86 , 3.87 , 3.86 , 3.53 , 3.26 , 3.21 , 3.05  and 2.03 . This agrees with previous reported NMR data of 6 (ref. m/z): [M]+ calcd. for C23H26NO7+, 428.1709; found, 428.1708.NMR data of f 6 ref. . HRMS : δ 10.06 , 7.09 , 6.36 , 6.17 , 5.85 , 5.88 , 4.14 , 3.85 , 3.82 , 2.90 , 2.37 , 2.32 , 2.21 , 2.05 , 1.99 . 13C-NMR : δ 191.74, 170.73, 153.22, 150.98, 148.03, 138.77, 133.67, 133.11, 131.28, 131.04, 125.42, 116.86, 116.15, 102.04, 101.11, 76.09, 62.27, 62.22, 56.49, 51.45, 45.76, 28.95 and 21.30. HRMS (m/z): [M+H]+ calcd. for C23H26NO8+, 444.1658; found, 444.1655.NMR data of 9 were as follows: 1H-NMR : δ 7.22 , 6.31 , 5.99 , 5.95 , 5.67 , 4.31 , 3.87 , 3.79 , 2.43 , 2.43 , 2.36 , 2.21 , 1.80 . This agrees with previous reported NMR data of 9 (ref. m/z): [M+H]+ calcd. for C21H22NO7+, 400.1396; found, 400.1394.NMR data of f 9 ref. , 73. HRM1 were as follows: 1H-NMR : δ 7.27 (1H), 6.52 (1H), 6.17 (1H), 6.00 (2H), 5.54 (1H), 4.23 (1H), 3.95 (3H), 3.86 (3H), 3.81 (3H), 2.43 (3H), 2.55 (1H), 2.43 (1H), 2.29 (1H) and 1.88 (1H). All the signals of 1 appear to be broad peaks in DMSO-d6, possibly due to chemical exchange. The chemical shifts and integration of peaks agree with previous reported NMR data of 1 (refs m/z): [M+H]+ calcd. for C22H24NO7+, 414.1553; found, 414.1554.NMR data of f 1 refs , 73. HRME. coli strain BL21(DE3) for PsMT2 or PsMT3 homodimer expression and PsMT2 and PsMT3 co-expression. The cells were cultured at 37 °C and 250 r.p.m. in 500 ml of lysogeny broth medium supplemented with 50 μg ml−1 kanamycin and/or 100 μg ml−1 ampicillin to a final OD600 between 0.4 and 0.6. The culture was then incubated on ice for 10 min before addition of 0.4 mM isopropylthio-β-D-galactoside to induce protein expression. The cells were further cultured at 16 °C for 12–16 h. The cells were harvested by centrifugation , resuspended in 25 ml lysis buffer , and lysed by sonication on ice. Cellular debris was removed by centrifugation , and appropriate affinity agarose resins were then added to the supernatant . The suspension was swirled at 4 °C for 2 h and loaded onto a gravity column.pCS3306 and pCS3307 were transformed individually and in combination into 2HPO4, 1.47 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, 0.1% Tween-20, 0002% sodium azide and pH 7.3). The T7-tagged protein was eluted with T7 Tag Elute Buffer , and each 1 ml of elute was neutralized with 150 μl of the T7 Tag Neutralization Buffer . For the purification of PsMT2/PsMT3 heterodimer, the supernatant of the cell lysate was first incubated with Ni-NTA agarose resin, and the Ni bound protein was isolated following the same procedure as that of PsMT2. The elution from the Ni-NTA agarose resin was concentrated and exchanged into 25 ml binding buffer, and incubated with T7 tag antibody agarose resin. The PsMT2/PsMT3 heterodimer was then purified following the same procedure for the purification of PsMT3. Purified enzymes were concentrated and exchanged into storage buffer  with the centriprep filters (Amicon), aliquoted and flash frozen using acetone/dry ice. Protein concentration was determined with the Bradford assayFor the purification of PsMT2, the PsMT2-bound resin was washed with 50 column volume of 10 mM imidazole in buffer A ), followed by 20 column volume of 20 mM imidazole in buffer A. PsMT2 was then eluted with 250 mM imidazole in buffer A. For the purification of PsMT3, the PsMT3-bound resin was washed with 10 column volume of binding buffer  of the purified PsMT2 homodimer, PsMT3 homodimer, PsMT2/PsMT3 heterodimer and PsMT3/Ps6OMT heterodimer were loaded on a NuPage Novex 4–12% Bis-Tris protein gel and run with NuPAGE MOPS SDS running buffer (Life Technologies) at 175 V for 45 min. The protein material was then semi-dry transferred to a nitrocellulose membrane or polyvinylidene difluoride membrane at 15 V for 15 min with transfer buffer (Life Technologies). The membrane was blocked with 5% bovine serum albumin in tris buffered saline and Tween-20 (TBST) buffer  for 1 h, and then probed overnight at 4 °C with Anti-6X His tag antibody  or Anti-T7 tag antibody  conjugated with HRP in TBST buffer for 12–16 h. Chemiluminescence was induced by SuperSignal West Pico substrate (Pierce) and images were acquired by a G:Box Chemi XT4 imaging system (Syngene).in vitro methyltransferase reactions were performed as previously described9 and 50 μg purified enzyme or no enzyme as a control. The reactions were performed overnight and quenched with 150 μl of 99.9% MeOH/0.1% formic acid. The quenched reaction mixture was analysed on an Agilent 6420 triple quad LC–MS  using positive ionization. Noscapine and narcotoline were detected with MRM using the highest characteristic precursor ion/product ion transition ; yeast alcohol dehydrogenase (150 kDa); bovine serum albumin (66 kDa); bovine erythrocytes carbonic anhydrase (29 kDa) and horse heart cytochrome c (12.4 kDa). Protein elution was detected by absorbance at 280 nm.The PsMT2/PsMT3 and PsMT2/Ps6OMT enzyme complex as a dimer were determined by SEC on a normal-phase HPLC (Agilent) using the BioSep-SEC-s4000 column. The column was equilibrated and eluted with 300 mM NaCl in 100 mM sodium phosphate buffer, pH 7.5, at a flow rate of 1 ml minThe authors declare that the data supporting the findings of this study are available within the article and its How to cite this article: Li, Y. et al. Engineering biosynthesis of the anticancer alkaloid noscapine in yeast. Nat. Commun. 7:12137 doi: 10.1038/ncomms12137 (2016).Supplementary Figures 1-14, Supplementary Tables 1-8 and Supplementary ReferencesS. cerevisiae strains and yeast expression constructs used in this study Engineered Plasmids used in this study DNA sequences of genes used in this study


It is well known that PUFA impede the LPS-mediated activation of the transcription factor NFkappaB. However, the underlying mode of action has not been clarified yet. To address this issue in a comprehensive approach, we used the monocyte/macrophage cell line RAW264.7 to investigate the consequences of a PUFA supplementation on the TLR4 pathway with a focus on (i) the gene expression of TLR4 itself as well as of its downstream mediators, (ii) the membrane microdomain localization of TLR4 and CD14, (iii) the stimulation-induced interaction of TLR4 and CD14. Our data indicate that the impairment of the TLR4-mediated cell activation by PUFA supplementation is not due to changes in gene expression of mediator proteins of the signaling cascade. Rather, our data provide evidence that the PUFA enrichment of macrophages affects the TLR4 pathway at the membrane level. PUFA incorporation into membrane lipids induces a reordering of membrane microdomains thereby affecting cellular signal transduction. It is important to note that this remodeling of macrophage rafts has no adverse effect on cell viability. Hence, microdomain disruption via macrophage PUFA supplementation has a potential as non-toxic strategy to attenuate inflammatory signaling. Pseudomonas aeruginosa, to the macrophage Toll-like receptor 4 (TLR4) activates the TLR4 signaling cascade, which eventually results in the activation of the nuclear factor kappa B (NFκB)  or the gram-negative bacterium B (NFκB) . The maiFκB) (κB . As a reFκB)  diactivity , and  unless noted otherwise. Cell culture flasks were purchased from Greiner Bio-One . HEPES (25 mmol/L)-buffered RPMI 1640 culture medium containing 300 mg/L L-glutamine was acquired from Pan-Biotech .2 cell culture flasks totaling 72 h at 37°C and 5% CO2 in a humidified atmosphere. Stimulation of cells was performed in the last 24 h of fatty acid supplementation by addition of LPS  or viable P. aeruginosa , bacterium/cell ratio 1:1). Periods of supplementation and stimulation were chosen in accordance with our previous investigations and were proven to result in a membrane fatty acid steady state as well as reproducible effects on macrophage functionality.The mouse monocyte/macrophage cell line RAW264.7 (ATCC number TIB-71) was used. RAW264.7 were cultured in RPMI 1640 medium containing 4.5 g/L glucose, 5% v/v FCS and 0.2% v/v ethanol (basic medium). The fatty acids alpha-linolenic acid , eicosapentaenoic acid , docosahexaenoic acid , linoleic acid  or arachidonic acid   were included in the culture medium in concentrations of 15 µmol/L using ethanol as a vehicle . Cells were cultured in the enriched media in 75 cmP. aeruginosa ATCC 10145 as described above. Gene expression was analyzed by a SYBR Green-based quantitative real-time PCR. Total RNA was extracted using the RNeasy kit  with an on-column DNase digestion. 1 µg RNA was reverse transcribed to cDNA using Oligo(dT)12−18Primers and SuperScript® III Reverse Transcriptase , and quantitative real-time PCR was performed by means of suitable murine RT2 qPCR primer assays  and the SensiMix™ SYBR Kit . Genes of interest amplified are TLR4, CD14, MyD88, IRAK-4 and TRAF6; housekeeping gene amplified is CASC3. Positive controls  as well as negative controls  were performed in each run. Thermal cycling was carried out on the Rotor-Gene 6000 real-time PCR system  at 95°C for 10 min followed by 45 cycles of 95°C for 15 s, 55°C for 30 s and 72°C for 15 s. Relative quantification was performed with the Rotor-Gene 6000 Series Software 1.7. The quantitative real-time PCR was performed in triplicate of three independent RNA isolations.RAW264.7 were cultured, supplemented and stimulated with LPS or P. aeruginosa as described above. The cells were fixed in 4% paraformaldehyde for 10 min, rinsed in PBS two times and permeabilized in 0.1% Triton-X100 in PBS for 5 min. Non-specific binding sites were blocked by incubation in 1% BSA in PBS for 1 h. Afterwards cells were incubated in a humidified chamber for 1 h at room temperature with species-specific primary antibodies against GM1, TLR4  as well as CD14 , rinsed in PBS three times and incubated for 1 h in a humidified chamber at room temperature in the dark with appropriate secondary antibodies conjugated to Alexa Fluor 350, Alexa Fluor 488 and Alexa Fluor 647 respectively . The coverslips with the labeled cells were rinsed in PBS three times, mounted onto glass slides and examined under a BZ-9000 fluorescence microscope . Microscopic images were analyzed using the public domain software ImageJ (Version 1.47n). Co-localization was quantified using Manders’ coefficient as previously described  in presence of the protease inhibitor cocktail Mammalian Protease Arrest  in a final concentration of 0.2% v/v. Cell lysates with a total protein concentration of 100 µg/mL were incubated with 1 µg of an anti-mouse CD14 antibody  at 4°C for 1 h. Immune complexes were captured with 20 µl Protein A/G Plus–Agarose beads  at 4°C overnight and under constant rotation. Agarose beads were washed 3 times with lysis buffer, resuspended in 35 µl Roti-Load 1 sample buffer  and boiled for 10 min. Immunoprecipitated proteins were separated by SDS-PAGE using the Amersham ECL Gel box and the Amersham ECL Gel 4–12% , and subsequently blotted on a 0.22 µm nitrocellulose membrane  for Western blot analysis. The membrane was blocked in PBS containing 0.05% Tween20 and 5% nonfat dry milk and divided into two parts . Immunostaining was performed overnight at 4°C using appropriated species-specific primary antibodies against TLR4  and CD14 , followed by a 2 h incubation at room temperature with appropriate horseradish peroxidase conjugated secondary antibodies . Bands were visualized using the Clarity ECL western blotting substrate  according to the manufacturer’s instructions. Band specificity was verified by performing IgG controls as well as by checking protein size using the PageRuler prestained protein ladder . Band intensities were determined densitometrically using the GeneTools image analysis software from Syngene . Four independent experiments were performed in duplicate for each combination of PUFA supplementation and stimulation of cells.RAW264.7 were cultured, supplemented and stimulated with LPS or t test was used to identify significant differences between means. The statistical analysis was carried out by means of the program GraphPad Prism 6 . In all cases, p < 0.05 was considered to indicate significant differences.Data are shown as mean ± standard deviation (S.D.). Two-way analysis of variance followed by unpaired Students κB, however, the underlying mechanisms have not been elucidated yet. Nevertheless, according to scientific literature there are two possible scenarios. On the one hand unsaturated fatty acids are discussed to diminish the gene expression of the TLR4 receptor (P. aeruginosa ATCC 10145 as stimulus. Supplementation and stimulation protocols were in accordance with previous investigations of the working group. A supplementation period of 72 h is needed to complete the incorporation of the fatty acids into the membrane of RAW264.7 cells. A stimulation period of 24 h is proven to result in macrophage (M1) activation, which is characterized by an increase in reactive oxygen intermediates  coefficient used for analyzing the microscopic data is not sensitive to differences in pixel intensities of an image . For thiThe interaction of TLR4 with its co-receptor CD14 being a prerequisite for the initiation of the TLR4 signaling cascade , was detThere was a clear trend towards an increased association of TLR4 with CD14 due to stimulation  and 4B. P. aeruginosa all PUFA tested with the exception of AA were found to interfere with receptor-co-receptor formation  Cytokine production of cells stimulated with LPS (1 µg/mL) for 24 h. (B) Cytokine production of cells supplemented with alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) in a concentration of 15 µM for 72 h. (C) Cytokine production of cells supplemented with alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) in a concentration of 15 µM for 72 h, and stimulated with LPS (1 µg/mL) in the last 24 h of supplementation. Data are mean ± S.D. (N = 6). Asterisks indicate a statistically significant difference compared with the unstimulated or unsupplemented controls . For further details please refer to RAW264.7 macrophages were cultured in basic medium . The pro-inflammatory cytokines IL-1Click here for additional data file.10.7717/peerj.1663/supp-2Figure S2GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-3Figure S3GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-4Figure S4GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-5Figure S5GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-6Figure S6GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-7Figure S7GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-8Figure S8GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-9Figure S9GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-10Figure S10GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-11Figure S11GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-12Figure S12GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-13Figure S13GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-14Figure S14GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-15Figure S15GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-16Figure S16GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-17Figure S17GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-18Figure S18GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-19Figure S19GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-20Figure S20GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-21Figure S21GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-22Figure S22GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-23Figure S23GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-24Figure S24GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-25Figure S25GM1 is labeled in red; TLR4 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-26Figure S26GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-27Figure S27GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-28Figure S28GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-29Figure S29GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-30Figure S30GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.10.7717/peerj.1663/supp-31Figure S31GM1 is labeled in red; CD14 is labeled in green. Scale bar represents 20 µm. Related to Click here for additional data file.


Mitral regurgitation (MR) is common in patients with ischemic cardiomyopathy (ICM) and independently associated with worse mortality. We sought to determine the impact of adverse LV remodeling, total myocardial infarct (MI) size, location and extent of regional MI, and mitral valve geometry on the severity of mitral regurgitation.A total of 494 patients with LVEF ≤ 40% and ≥ 70% stenosis in ≥ 1 epicardial coronary artery and prior history of CABG or PCI were screened. Patients with transthoracic echocardiogram and CMR studies within 7 days were selected. Forty-two patients (9%) were excluded either due to prior mitral valve surgery, unavailable studies or organic mitral valve disease. Other 42 patients were excluded because of no mitral regurgitation noted on their study. Mitral regurgitation was assessed using semi-quantitative vena contracta method from a parasternal long axis zoomed view obtained of the mitral valve. LV volumes and EF were calculated from cine short axis CMR images. Mitral valve geometry was measured on end-systolic 2, 3, and 4 chamber cine views. LV scar was measured 10-15 minutes after gadolinium injection and using phase-sensitive inversion recovery sequence.The mean age of the cohort n = 410) was 63 ± 11 years and males represented 75%. LVEF, LV end-systolic volume index, mean total infarct size, MV annulus index, MV tenting area index, and MV displacement index were: 23 ± 0.5%, 115 ± 2 ml/m2, 31 ± 17%, 1.7 ± 0.5 cm2/m2, 0.88 ± 0.48 cm2/m2, respectively. Mean inferior, lateral, anterior infarct size were: 27 ± 21%, 25 ± 20%, 37 ± 23%. MR was classified as: mild (63%), moderate (22%) or severe (15%). The mean vena contracta was 0.33 ± 0.24 cm. Multiple variable linear regression (R2 = 0.23) revealed that male gender, worsening GFR, increase extent of inferior LV scar, LV ESVi and MV tenting area index were independent predictors of MR severity, whereas overall scar extent, anterior or lateral location were not. (Figure 0 was 63 Global LV remodeling, regional inferior myocardial scarring, and mitral valve geometry are more important predictors of worsening MR than total infarct size in patients with advanced ischemic cardiomyopathy.None.


Algoriphagus sp. strain M8-2 was isolated from a brackish lake, Lake Sanaru, in Hamamatsu, Japan, as a filterable bacterium through a 0.22-µm-pore-size membrane filter. We report here the complete nucleotide sequence of the M8-2 genome . Algoriphagus sp. strain M8-2 was isolated from a blackish lake, Lake Sanaru, in Hamamatsu, Shizuoka, Japan . This strain was found and isolated on 28 April 2014 as one of the filterable bacteria from the lake. The strain M8-2 can form colonies on an agar plate even after filtration of the lake water through a 0.22-µm-pore-size membrane filter. It can grow on Difco marine broth 2216 with 1.5% agar at 30°C. The colony morphology on the medium was a circular form and convex elevation with a pink-orange pigment.in silico analyses and PCR and Sanger sequencing of the PCR products. The finished sequence was confirmed by FinishChecker, which is an accessory tool of GenoFinisher.The genome of M8-2 was determined using MiSeq technology with a paired-end library  prepared using the TruSeq DNA PCR-free library preparation kit (Illumina). A total of 2.0 million reads (479 Mb) preprocessed by ShortReadManager, which corresponds to a depth of 126-fold genome coverage, were used for assembly by the Newbler version 2.8 software , and gaphttp://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html), and the resulting annotation was manually inspected with respect to the start codon positions using the Microbial Genome Annotation Pipeline (http://www.migap.org/), Prokka  (DDBJ/EMBL/GenBank accession no. BBFN00000000), A. machipongonensis (4.89 Mb) (A. mannitolivorans (4.15 Mb)  (A. terrigena (5.78 Mb) (A. vanfongensis (4.71 Mb) . Sequence annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (erans 4.8 Mb (DDBJCP012836.The nucleotide sequence of the M8-2 genome was deposited in the DDBJ/EMBL/GenBank databases under accession no.


Anthranilic acid (ANA) and 3-hydroxyanthranilic acid (3-HANA) are kynurenine pathway intermediates of the tryptophan metabolism. A hitherto unemployed method combination, differential pulse voltammetry, mass spectrometry (nano-ESI–MS), deoxyribose degradation and iron(II) autoxidation assays has been employed for studying of their redox chemistry and their interactions with iron(II) and iron(III) ions. Both acids inhibited the Fenton reaction by iron chelation and ROS scavenging in the deoxyribose degradation assay. In the iron(II) autoxidation assay, anthranilic acid showed antioxidant effects, whereas 3-hydroxyanthranilic acid exhibited apparent pro-oxidant activity. The differential pulse voltammograms of free metabolites and their iron(II) coordination complexes reflected these properties. Nano-ESI–MS confirmed ANA and 3-HANA as efficient iron(II) chelators, both of which form coordination complexes of ligand:iron(II) ratio 1:1, 2:1, and 3:1. In addition, nano-ESI–MS analyses of the oxidation effects by hydroxyl radical attack identified 3-HANA as strikingly more susceptible than ANA. 3-HANA susceptibility to oxidation may explain its decreased concentrations in the reaction mixture. The presented observations can add to explaining why 3-HANA levels decrease in patients with some neurological and other diseases which can often associated with elevated concentrations of ROS. Anthranilic and 3-hydroxyanthranilic acid are tryptophan metabolites. Their redox chemistry and iron chelation were investigated. •Anthranilic (ANA) and 3-hydroxyanthranilic acid (3-HANA) are tryptophan metabolites.•ANA and 3-HANA levels change in patients suffering from some neurological diseases.•Oxidative hydroxyl radical attack affects 3-HANA more than ANA.•Both acids can serve as ligands of transition metals and affect their redox cycling.•These findings might explain why 3-HANA levels decrease in these patients. Anthranilic acid  and 3-hydroxyanthranilic acid (3-HANA)  are metaDuring the last decades, ANA and 3-HANA have been recognized to affect brain and immune system function. Patients with neurological disorders usually show marked changes in the levels of these two tryptophan metabolites Although the redox and chelation properties of both acids have been investigated extensively in the last decades, some studies demonstrated controversial results. This stimulated us to explore these two specific tryptophan metabolites in more detail by employing a set of methods that have not yet been used in this combination in previous studies: differential pulse voltammetry, and direct infusion mass spectrometry (nano-ESI–MS), deoxyribose degradation and iron(II) autoxidation assays.All chemicals used were purchased from Sigma–Aldrich . Water had Milli-Q quality.−1, step potential was 5.25 mV, modulation amplitude was 25 mV, modulation time was 50 ms, and interval time was 250 ms. Scan potential was from −500 to +1200 mV. FeSO4 was dissolved in degassed water at a concentration 10 mM. The stock solutions of the measured acids were prepared by dissolving in degassed buffer (0.1 M phosphate buffer pH 7.4). The ionic strength of the buffer was 1 M and it was adjusted by K2SO4. The samples for the electrochemical measurements of the acids were prepared by mixing of 1 mL their stock solution with 8 mL of the buffer and 1 mL of water. The final concentration of the acid was 1 mM. The samples for the coordination complex analysis were prepared by mixing 1 mL of aqueous FeSO4 solution with 9 mL of the degassed buffer or buffer–anthranilic or 3-HANA solution. The final applied concentration ratio of acid:Fe was 4:1. The solutions of electrolytes were degassed by argon for 10 min and measurements were carried out under argon atmosphere at a room temperature.Voltammetric curves were recorded in a three-electrode system, μAutolab PGSTAT type III . The working electrode was a glassy carbon electrode of 3 mm diameter, an Ag/AgCl (saturated KCl) electrode was used as reference, and platinum wire as a counter electrode. The glassy carbon electrode was washed with water and then polished by aluminium oxide powder (0.3 μm of grain size) before every measurement. The effective scan rate of the voltammetry was 21 mV s6 ions with a resolution of 100,000 in the m/z range of 80–2000. In order to achieve even higher mass accuracy, a lock mass option was enabled and the cyclomethicone N5 ions generated in the electrospray process from ambient air (m/z = 371.101230) were used for internal recalibration in real time. This allowed mass accuracies of <1 ppm. Specific tune settings for the MS were as follows: spray voltage was set to 1.8 kV; capillary voltage was 45 V, tube lens offset 150 V and capillary temperature was set at 180 °C, no sheath gas and auxiliary gas were used. ANA and 3-HANA coordination complexes were measured according to a modified method that was published previously by Sarowar et al. II complexes were prepared from a 1 mM stock solution of ANA or 3-HANA in degassed methanol by adding of appropriate amount of degassed aqueous FeCl2 solution (500 μM) in the (ANA or 3-HANA: Fe) molar ratios 1:1, 2:1, 3:1, and 4:1. Before the measurement, the samples were diluted 1:10 or 1:100 with water/methanol  mixture. For the nano-ESI–MS measurement, gold coated glass emitters  with 5 μL of this final sample were used. The samples for nano-ESI–MS analyses of anthranilic and 3-hydroxyanthranilic acid oxidation stability were prepared as followed: The tested compound was dissolved in aqueous ammonium bicarbonate buffer solution  to 1 mM solution. To 500 μL of this solution, 300 μL of 10 mM aqueous ammonium bicarbonate buffer, 100 μL of degassed aqueous FeCl2 solution (100 μM) and 100 μL of aqueous H2O2 solution (55 mM) were added. Standard 1.5 mL sample vials  were used as reaction vials. The reaction mixtures was incubated at 27 °C and analyzed in reaction times 3 and 8 h. For the nano-ESI–MS measurement, 50 μL of the reaction mixture was diluted with 450 μL of 0.2% (v/v) formic acid in methanol and glass emitters  filled with 5 μL of this mixture were used. The isotope exact masses of the complex ions and all other designated ions were calculated with ChemDoodle 7.0.2, iChemLabs, LLC, Somset, NY.MS analyses were performed out on a Thermo Electron LTQ-Orbitrap XL mass spectrometer equipped with a nano electrospray ion source  and operated under Xcalibur software, in the positive ionization mode. The instrument was calibrated using the manufacturer's calibration standards. The Fourier transformed full scan mass spectra were acquired at a target value of 102PO4/KOH buffer solution  and diluted serially; to 125 μL of this solution, 25 μL of a 10.4 mM 2-deoxy-d-ribose solution in the same buffer system and 50 μL of FeCl3 (FeCl3 variant) or FeIIIEDTA (FeIIIEDTA variant) solution (50 μM) were added. The complex of FeIII with EDTA was prepared separately; the 104 μM EDTA solution in the buffer was premixed with the aqueous 100 μM FeCl3 solution . Further, 25 μL of 10.0 mM aqueous solution H2O2 and 25 μL of 1.0 mM ascorbic acid in the buffer were added to start the Fenton reaction in the H2O2/FeIII/ascorbic acid reaction mixture. In the other deoxyribose degradation assay systems, H2O2 or ascorbic acid was replaced by the same volume of water or buffer, respectively. The final concentrations of the tested acids were 2–500 μM. Thiobarbituric acid reactive species (TBARS) were determined photometrically at 532 nm after reaction with thiobarbituric acid and subsequent extraction of the pink pigment with 1-butanol. The H2O2/FeIII/ascorbic acid reaction mixture served as positive control and represented 100% TBARS detection in all variants and also served as comparative standard for each experiment. Blanks contained the full reaction mixtures except for 2-deoxy-d-ribose and were determined in each experiment. Experiments were performed in triplicates. The temperature during incubation was 27 °C. Variants containing H2O2 were evaluated after 1 h; variants without H2O2 were evaluated after 16 h incubation.The deoxyribose degradation assay and its various variants follow published procedures 2PO4/KOH buffer solution  and diluted serially; to 125 μL of this solution, 25 μL of a 52 mM 2-deoxy-d-ribose solution in the same buffer system, 50 μL of the buffer, and 50 μL of degassed aqueous FeSO4 solution (50 μM) were added. The final concentrations of the tested compounds were 2–500 μM. Blanks contained the full reaction mixtures except for 2-deoxy-d-ribose. Standard 1.5 mL sample vials  were used as reaction vials. The mixtures were incubated at 27 °C for 16 h. Thereafter, 250 μL of 1.0% thiobarbituric acid dissolved in 3% trichloroacetic acid (w/v) was added to each vial to detect TBARS. The vials were heated in a water bath at 80 °C for 30 min. The reaction was stopped by transferring the vials into an ice water bath for 3 min. To extract the TBARS, 600 μL of 1-butanol was added, and the mixture was rigorously vortexed. The butanol layers of the vials, each 350 μL, were pipetted into flat bottomed 96 well plates , and the absorbance was determined with a microplate reader  at 532 nm. Experiments were performed in triplicate. Reaction mixtures lacking the test compound served as the positive control (100% TBARS). The phosphate buffer and water, which were used as solvents for the tested substances or FeSO4, were degassed by argon for 10 min at least.The assay was carried out according procedures published elsewhere post hoc test.Statgraphics Centurion XVI  was used to perform analyses of variance (ANOVA) employing 95% Duncan's multiple range Ortho-aminophenols, such as 3-HANA, usually exhibit redox activity because of their easy oxidation to quinone imines The differential pulse voltammogram (DPV) of ANA showed one peak at 766 mV (peak 1), corresponding to oxidation of the amino group a, and 3-In addition, ANA and 3-HANA were investigated for their possible capability of forming coordination complexes with iron(II). The voltammogram demonstrated that iron(II) ions formed various coordination complexes with phosphate (buffer component), which resulted in a broad dominant peak with two maxima at the potentials at −235 mV (peak maximum 1) and −18 mV (peak maximum 2) b. When i+, m/z 138.0551  and m/z 160.0370 [M + Na]+ .Consequently, coordination complex formation of ANA and 3-HANA with iron(II) was explored by nano-ESI–MS . This mem/z 224.0005  corresponds to a coordination complex ion with one ANA ligand [M−H + FeII + CH3OH]+. The following iron isotopes were detectable: C8H1054FeNO3, m/z 222.0053, calc. m/z 222.0052, rel. int. 6%, calc. rel. int., 6%; C8H1056FeNO3, rel. int. 100%, calc. rel. int. 100%; C8H1057FeNO3, m/z 225.0039, calc. m/z 225.0038, rel. int. 7%, calc. rel. int. 9%; C8H1058FeNO3, m/z 226.0044, calc. m/z 226.0043, rel. int. 0.2%, calc. relative int. 0.2%.An ion detected at m/z 329.0219 that corresponds to [2M−H + FeII]+, C14H1356FeN2O4, calc. m/z 329.0219. The following iron isotope ions were detectable: C14H1354FeN2O4, 327.0267, calc. 327.0266, rel. int. 6%, calc. rel. int. 6%; C14H1356FeN2O4, rel. int. 100%, calc. rel. int. 100%; C14H1357FeN2O4, m/z 330.0254, calc. rel. int. 330.0252, rel. int. 14%, calc. rel. int. 15%; C14H1358FeN2O4, m/z 331.0263, calc. m/z 331.0262, rel. int. 0.3%, calc. rel. int. 0.3%.A further metal complex ions was detected at m/z 351.0039  represented the Na adduct of the former complex [2M–2H + FeII + Na]+. The following iron isotope ions were detectable: C14H1254FeN2NaO4, m/z 349.0087, calc. m/z 349.0085, rel. int. 5%, calc. int. 6%; C14H1256FeN2NaO4, rel. int. 100%, calc. int. 100%; C14H1257FeN2NaO4, m/z 352.0071, calc. m/z 352.0073, rel. int. 14%, calc. rel. int. 15%, C14H1258FeN2NaO4, m/z 353.0055, calc. m/z 353.0057, rel. int. 0.3%, calc. rel. int. 0.3%.An m/z 466.0694 was assigned to C21H2056FeN3O6, [3M−H + FeII]+, calc. m/z 466.0693. The following iron isotope ions were detectable: C21H2054FeN3O6, m/z 464.0742, calc. m/z 464.0739, rel. int. 4%, calc. rel. int. 6%; C21H2056FeN3O6, rel. int. 100%, calc. rel. int. 100%; C21H2057FeN3O6, m/z 467.0726, calc. m/z 467.0727, rel. .int. 20%, calc. rel. int. 22%; C21H2058FeN3O6, m/z 468.0734, calc. m/z 468.0732, calc. rel. int. 0.5%.A further ion detected at m/z 154.0499 was identified as the protonated ion of 3-HANA [M + H]+, calc. 154.0500. (+) appeared as m/z 176.0319 signal .In the ESI–MS spectrum of iron(II)−3-HANA solution, a prominent peak at 54.0500. b. The 3-II + CH3OH]+, C8H1056FeNO4, m/z 239.9955, calc. m/z 239.9955. The following iron isotope ions were detectable: C8H1054FeNO4, m/z 238.0002, calc. m/z 238.0000, rel. int. 6%, calc. rel. int. 6%; C8H1056FeNO4, rel. int. 100%, calc. rel. int. 100%; C8H1057FeNO4, m/z 240.9988, calc. m/z 240.9987, rel. int. 8%, calc. rel. int. 8%); C8H1058FeNO4, m/z 241.9994, calc. m/z 241.9996, rel. int. 0.5%, calc. rel. int. 0.8%.The first metal complex was assigned to the ion [M−H + Fem/z 361.0117 that correspond to [2M−H + FeII]+, C14H1356FeN2O6, calc. m/z 361.0117. The following iron isotope ions were detectable: C14H1354FeN2O6, m/z 359.0165, calc. m/z 359.0164, rel. int. 6%, calc. rel. int. 6%; C14H1356FeN2O6, rel. int. 100%, calc. rel. int. 100%; C14H1357FeN2O6, m/z 362.0151, calc. m/z 362.0150, rel. int. 15%, calc. rel. int. 15%; C14H1358FeN2O6, m/z 363.0189, calc. m/z 363.0185, rel. int. 0.7%, calc. rel. int. 1%.A further metal complex ion with two 3-HANA ligands had an m/z 514.0541, corresponding to C21H2056FeN3O9, [3M−H + FeII]+, calc. m/z 514.0541. The following iron isotope ions were detectable: C21H2054FeN3O9, m/z 512.0588, calc. m/z 512.0588, rel. int. 3%, calc. int. 6%; C21H2056FeN3O9, rel. int. 100%, calc. rel. int. 100%; C21H2057FeN3O9, m/z 515.0576, calc. m/z 515.0574, rel. int. 18%, calc. rel. int. 22%; C21H2058FeN3O9, m/z 516.0578, calc. m/z 516.0583, rel. int. 2%, calc. rel. int. 2%.A metal complex ion with three 3-HANA ligands was identified at d-ribose by hydroxyl radicals arising in the Fenton reaction d-ribose leads to the production of thiobarbituric acid reactive species (TBARS). The reaction mixture contains hydrogen peroxide, iron(III) and ascorbic acid. Ascorbic acid starts the Fenton reaction by reduction of iron(III) to iron(II). Iron is added either as FeCl3 or in complex with ethylenediaminetetraacetic acid (EDTA). Ascorbic acid is used because it is known as a common cellular reducing agent. If iron ions are added as FeCl3, they can form complexes with the test compound, as EDTA complex this is less likely to occur. Further modifications of this assay, for example if hydrogen peroxide and/or ascorbic acid are not added, provide additional information about the redox chemistry of the test compounds All ions mentioned above were detected with different intensities in ANA or 3-HANA: Fe molar ratios 1:1, 2:1, 3:1 and 4:1 (data not shown). These observations were corroborated by the deoxyribose degradation assay results . The pri2O2/FeIII/ascorbic acid and FeIII/ascorbic acid deoxyribose degradation assay variants (III or H2O2/FeIII assay variants (data not shown). In contrast, Iwahashi et al. observed that 3-HANA increased hydroxyl radical production in reaction mixtures containing iron(III) and hydrogen peroxide or superoxide dismutase; the concentrations of hydroxyl radicals were determined by EPR (electron paramagnetic resonance) d-ribose such as it is the case in this study. In the iron(III) reduction assay, they employed a spectrophotometric determination of iron(II) concentrations by iron(II) chelators, bathophenanthroline disulfonic acid or phenanthroline ANA and 3-HANA proved as efficient antioxidants in the Hvariants a–d. The d-ribose decomposition products). In this assay, iron does not require to be reduced before participation in the Fenton reaction To clarify the contradictions, we performed an iron(II) autoxidation assay Both mechanisms can contribute to the antioxidant activity of ANA. If iron is a central atom in coordination complexes with ANA, the redox potential of iron is shifted into the anodic direction as shown by differential pulse voltammetry. An anodic shift decreases the rate of iron(II) autoxidation. Furthermore, the 2-amino group can also contribute to the antioxidant effect of ANA by hydroxyl radical scavenging.The pro-oxidant effect of 3-HANA in the iron(II) autoxidation assay can be explained by semiquinone formation. The hydroxyl group of 3-HANA can be oxidized to a semiquinone by superoxide anion radical, a reaction product of iron(II) autoxidation. Semiquinones and their derivatives are potent reducing agents which can reduce molecular oxygen to superoxide anion radical The nano-ESI–MS analyses of ANA and 3-HANA oxidation stability confirmed higher redox reactivity of the latter compared to the former . The expChanging ANA and 3-HANA concentrations as well as their actual ratio in tissues can modulate T-cell activity in the immune system. 3-HANA can also affect various inflammatory factors Both, ANA and 3-HANA, are redox active and can act as antioxidants in certain chemical environments. Furthermore they form coordination complexes with iron. A combination of differential pulse voltammetry, deoxyribose degradation assay, iron(II)-autoxidation assay and direct infusion ESI–MS of coordination complexes and oxidation reaction solutions succeeded in revealing some minor but important differences in the redox chemistry of these two tryptophan metabolites. 3-HANA is much more instable than ANA when attacked by hydroxyl radicals in an oxidative stress scenario. The high redox reactivity of 3-HANA, especially in presence of iron(II), concurs with the observation that patients suffering from various neurological diseases  show decreased levels of this metabolite.