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Toncarine | c1(C)ccc2oc(ccc2c1)=O | Xanthotoxin, herniarin, c1(C)ccc2oc(ccc2c1)=O, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Bergamottin | C(C/C(C)=C/COc1c2ccc(oc2cc2c1cco2)=O)C=C(C)C | Xanthotoxin, herniarin, toncarine, C(C/C(C)=C/COc1c2ccc(oc2cc2c1cco2)=O)C=C(C)C, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′epoxyC(C/C(C)=C/COc1c2ccc(oc2cc2c1cco2)=O)C=C(C)C (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Oxypeucedanin | c12cc3occc3c(c2ccc(=O)o1)OCC1OC1(C)C | Xanthotoxin, herniarin, toncarine, bergamottin, c12cc3occc3c(c2ccc(=O)o1)OCC1OC1(C)C, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Psoralen | C1C2OC=CC=2C=C2C=1OC(=O)C=C2 | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, C1C2OC=CC=2C=C2C=1OC(=O)C=C2, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Isopimpinellin | C1(=C2C=COC2=C(OC)C2OC(=O)C=CC=21)OC | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, C1(=C2C=COC2=C(OC)C2OC(=O)C=CC=21)OC, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Bergapten | o1ccc2c1cc1c(c2OC)ccc(o1)=O | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, o1ccc2c1cc1c(c2OC)ccc(o1)=O, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Trioxsalen | c12cc3c(c(C)c1oc(C)c2)oc(cc3C)=O | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). c12cc3c(c(C)c1oc(C)c2)oc(cc3C)=O and 6′,7′epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Citropten | c1c(OC)cc2c(ccc(=O)o2)c1OC | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). c1c(OC)cc2c(ccc(=O)o2)c1OC (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Methanol | CO | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade CO, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in CO. | 252558909 |
Herniarin | c1c(oc2cc(ccc2c1)OC)=O | Xanthotoxin, c1c(oc2cc(ccc2c1)OC)=O, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Toncarine | O=c1oc2c(cc(cc2)C)cc1 | Xanthotoxin, herniarin, O=c1oc2c(cc(cc2)C)cc1, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Bergamottin | C(/C=C(\C)CCC=C(C)C)OC1C2C=COC=2C=C2C=1C=CC(O2)=O | Xanthotoxin, herniarin, toncarine, C(/C=C(\C)CCC=C(C)C)OC1C2C=COC=2C=C2C=1C=CC(O2)=O, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxyC(/C=C(\C)CCC=C(C)C)OC1C2C=COC=2C=C2C=1C=CC(O2)=O (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Oxypeucedanin | O1C(C)(C)C1COc1c2ccc(=O)oc2cc2c1cco2 | Xanthotoxin, herniarin, toncarine, bergamottin, O1C(C)(C)C1COc1c2ccc(=O)oc2cc2c1cco2, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Psoralen | C12C(=CC3OC=CC=3C=1)OC(=O)C=C2 | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, C12C(=CC3OC=CC=3C=1)OC(=O)C=C2, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Isopimpinellin | C1(=C2C(C=CO2)=C(OC)C2=C1OC(C=C2)=O)OC | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, C1(=C2C(C=CO2)=C(OC)C2=C1OC(C=C2)=O)OC, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Bergapten | O=C1OC2C(=C(C3=C(OC=C3)C=2)OC)C=C1 | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, O=C1OC2C(=C(C3=C(OC=C3)C=2)OC)C=C1, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Trioxsalen | O=c1oc2c(C)c3c(cc(o3)C)cc2c(C)c1 | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). O=c1oc2c(C)c3c(cc(o3)C)cc2c(C)c1 and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Citropten | O(c1c2ccc(=O)oc2cc(OC)c1)C | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). O(c1c2ccc(=O)oc2cc(OC)c1)C (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. | 252558909 |
Methanol | CO | Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6 ,7 -epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade CO, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in CO. | 252558909 |
Methanol | CO | Citrus volatile oils from trusted suppliers were obtained from the collection of the Aromatic Plant Research Center (APRC, Lehi, UT, USA). A total of 374 cold-pressed Citrus oil samples from the APRC collection are listed in Table 4. A simple dilute and shoot technique (1 µL oil in 999 µL of CO) was used for sample preparation. Further dilution was performed whenever needed. | 252558909 |
Coumarins | C1C=C2C(=CC=1)OC(C(C(CC(C)=O)C1=CC=CC=C1)=C2O)=O | C1C=C2C(=CC=1)OC(C(C(CC(C)=O)C1=CC=CC=C1)=C2O)=O were quantified using a NEXERA UPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a mass spectrometer (Triple quadrupole, LCMS8060, Shimadzu, Kyoto, Japan). Target compounds were chromatographed on a Shimadzu Nexcol C 18 column (1.8 µm, 50 × 2.1 mm) with a C 18 guard column (Restek, Bellefonte, PA, USA) at 40 • C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The compounds were eluted using the following gradient: %10 B at 0 min, %20 B at 0.74 min, %60 B at 5.88 min, %90 B at 10 min, held at %100 B for 4 min, and %10 for 4 min before the next injection. The flow rate was maintained at 0.2 mL/min, and the injection volume was 1 µL. The UPLC system was connected to the MS by electrospray ionization (ESI) operating in positive ion mode. The interface, desolvation line, and heating block temperatures were 350, 250, and 400 • C, respectively. The capillary voltage was 4.5 kV, and CID gas was set at 350 kPa. Nebulizing gas flow was set at 3.0 L/min, and heating and drying gas were set at 10.0 L/min. The detection was completed in multiple reaction monitoring mode (MRM) ( Table 5). Samples were run in triplicates with external standards in between. Each run contained a quality control (QC) standard, and at least one QC standard was run at the beginning and the end of the run. The acquired chromatographic results were processed in LabSolutions Insight software version 3.2 (Shimadzu). For each compound, calibration curves (0.005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, and 0.1 ppm) were drawn by linking its peak area and its concentration. | 252558909 |
Methanol | CO | Coumarins were quantified using a NEXERA UPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a mass spectrometer (Triple quadrupole, LCMS8060, Shimadzu, Kyoto, Japan). Target compounds were chromatographed on a Shimadzu Nexcol C 18 column (1.8 µm, 50 × 2.1 mm) with a C 18 guard column (Restek, Bellefonte, PA, USA) at 40 • C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in CO (B). The compounds were eluted using the following gradient: %10 B at 0 min, %20 B at 0.74 min, %60 B at 5.88 min, %90 B at 10 min, held at %100 B for 4 min, and %10 for 4 min before the next injection. The flow rate was maintained at 0.2 mL/min, and the injection volume was 1 µL. The UPLC system was connected to the MS by electrospray ionization (ESI) operating in positive ion mode. The interface, desolvation line, and heating block temperatures were 350, 250, and 400 • C, respectively. The capillary voltage was 4.5 kV, and CID gas was set at 350 kPa. Nebulizing gas flow was set at 3.0 L/min, and heating and drying gas were set at 10.0 L/min. The detection was completed in multiple reaction monitoring mode (MRM) ( Table 5). Samples were run in triplicates with external standards in between. Each run contained a quality control (QC) standard, and at least one QC standard was run at the beginning and the end of the run. The acquired chromatographic results were processed in LabSolutions Insight software version 3.2 (Shimadzu). For each compound, calibration curves (0.005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, and 0.1 ppm) were drawn by linking its peak area and its concentration. | 252558909 |
Coumarins | C(CC(c1ccccc1)c1c(=O)oc2ccccc2c1O)(C)=O | Method validation was executed according to the USP<1225> Validation of compendial procedures [31] and ICH harmonized tripartite guideline validation of analytical procedures: text and methodology Q2(R1) [32]. Specificity, precision, accuracy, linearity, intermediate precision, and limit of quantification (LOQ) were determined using standard solutions. Distilled yuzu essential oil was used as a matrix (total C(CC(c1ccccc1)c1c(=O)oc2ccccc2c1O)(C)=O < 0.001 ppm). To prove the specificity of the method, standard solution mixtures and at least three blanks were processed to demonstrate the absence of interferences with the elution of the analytes. Precision and repeatability were determined by injecting six sample preparations spiked to a final concentration of 0.04 ppm and then calculating the RSD% between injections which may reach 10% for each. For the intermediate precision, the repeatability experiment was repeated on a second day and performed by a second analyst with the acceptance criterion of RSD ≤ 10 for each compound and each analyst. To determine the recoveries (accuracy) of the target compounds, three individually prepared samples of yuzu oil were spiked with three concentrations of the standard (LOQ, 0.04, and 0.05 ppm in triplicates). Recoveries were calculated by comparing the absolute peak areas with a reference measurement which must be within 80-120% of the expected value. Five concentrations from 0.001 to 0.1 ppm were used to determine linearity and a coefficient of determination (r) higher than 0.98 was needed. The data obtained during the linearity, precision, and accuracy studies were used to assess the range of the method for the target compounds. The acceptable range was defined as the concentration interval over which linearity, precision, and accuracy are acceptable. To estimate the LOQ, standard mixtures at low concentrations (0.0005 to 0.01 ppm) were analyzed. The calculated LOQ was determined using the signal-to-noise (S/N) ratio (10:1) and then injected 6 times. The acceptance criterion for the LOQ was RSD ≤ 15%. A calibration curve based on the linear range was prepared and injected to estimate the quantity of C(CC(c1ccccc1)c1c(=O)oc2ccccc2c1O)(C)=O in the oil samples. Additionally, QC standards at low (0.05 ppm) and high (0.1 ppm) concentrations were used. | 252558909 |
Coumarin | C1(=O)OC2C(C=C1)=CC=CC=2 | The average C1(=O)OC2C(C=C1)=CC=CC=2 concentrations (12 compounds) in the Citrus samples were used as variables in the multivariate analysis. First, the data matrix was standardized by subtracting the mean for each compound concentration and dividing it by the standard deviation. For the agglomerative hierarchical cluster (AHC) analysis, the 24 Citrus samples were treated as operational taxonomic units (OTUs). Pearson correlation was selected as a measure of similarity, and the unweighted pair group method with arithmetic average (UPGMA) was used for cluster definition. Principal component analysis (PCA) was performed for the visual comparison of the C1(=O)OC2C(C=C1)=CC=CC=2 compositions of the different Citrus groups using the 12 C1(=O)OC2C(C=C1)=CC=CC=2 components as variables, with a Pearson correlation matrix. The AHC and PCA analyses were performed using XLSTAT v. 2018.1.1.62926 (Addinsoft, Paris, France). | 252558909 |
Coumarins | o1c(=O)c(C(c2ccccc2)CC(C)=O)c(c2c1cccc2)O | In this study, we developed and validated a simple and sensitive UPLC-MS/MS method for the detection and quantification of 14 selected oxygen heterocyclic compounds (o1c(=O)c(C(c2ccccc2)CC(C)=O)c(c2c1cccc2)O and furanoo1c(=O)c(C(c2ccccc2)CC(C)=O)c(c2c1cccc2)O). Targeted screening using this method was successfully completed for the essential oils of 12 different Citrus species. To our knowledge, this is the most comprehensive investigation of coumarin and furanocoumarin profiles across commercial-scale Citrus oils to date. The lowest amount was detected in calamansi oil. Expressed oil of Italian bergamot showed the highest furanocoumarin content and the highest level of any individual furanocoumarin (bergamottin). Remarkable differences were observed in the coumarin and furanocoumarin levels among oils of different crop varieties and origins within the same species. We found potential correlations between bergapten and xanthotoxin which matches with known biosynthetic pathways. Patterns in furanocoumarin profiles lined up with known variations among the Citrus ancestral taxa. Using multivariate analysis, we were able to divide the Citrus oils into 5 main groups (bergamots; lime and German lemon; yuzu and grapefruit; oranges, tangerines, clementines, mandarins, calamansi, and petitgrains; and lemons) and correlate them to the coumarin compositions. | 252558909 |
Coumarin | c1(=O)ccc2ccccc2o1 | In this study, we developed and validated a simple and sensitive UPLC-MS/MS method for the detection and quantification of 14 selected oxygen heterocyclic compounds (c1(=O)ccc2ccccc2o1s and furanoc1(=O)ccc2ccccc2o1s). Targeted screening using this method was successfully completed for the essential oils of 12 different Citrus species. To our knowledge, this is the most comprehensive investigation of c1(=O)ccc2ccccc2o1 and furanoc1(=O)ccc2ccccc2o1 profiles across commercial-scale Citrus oils to date. The lowest amount was detected in calamansi oil. Expressed oil of Italian bergamot showed the highest furanoc1(=O)ccc2ccccc2o1 content and the highest level of any individual furanoc1(=O)ccc2ccccc2o1 (bergamottin). Remarkable differences were observed in the c1(=O)ccc2ccccc2o1 and furanoc1(=O)ccc2ccccc2o1 levels among oils of different crop varieties and origins within the same species. We found potential correlations between bergapten and xanthotoxin which matches with known biosynthetic pathways. Patterns in furanoc1(=O)ccc2ccccc2o1 profiles lined up with known variations among the Citrus ancestral taxa. Using multivariate analysis, we were able to divide the Citrus oils into 5 main groups (bergamots; lime and German lemon; yuzu and grapefruit; oranges, tangerines, clementines, mandarins, calamansi, and petitgrains; and lemons) and correlate them to the c1(=O)ccc2ccccc2o1 compositions. | 252558909 |
Bergamottin | O1C2C(=C(C3=C(OC=C3)C=2)OC/C=C(\C)CCC=C(C)C)C=CC1=O | In this study, we developed and validated a simple and sensitive UPLC-MS/MS method for the detection and quantification of 14 selected oxygen heterocyclic compounds (coumarins and furanocoumarins). Targeted screening using this method was successfully completed for the essential oils of 12 different Citrus species. To our knowledge, this is the most comprehensive investigation of coumarin and furanocoumarin profiles across commercial-scale Citrus oils to date. The lowest amount was detected in calamansi oil. Expressed oil of Italian bergamot showed the highest furanocoumarin content and the highest level of any individual furanocoumarin (O1C2C(=C(C3=C(OC=C3)C=2)OC/C=C(\C)CCC=C(C)C)C=CC1=O). Remarkable differences were observed in the coumarin and furanocoumarin levels among oils of different crop varieties and origins within the same species. We found potential correlations between bergapten and xanthotoxin which matches with known biosynthetic pathways. Patterns in furanocoumarin profiles lined up with known variations among the Citrus ancestral taxa. Using multivariate analysis, we were able to divide the Citrus oils into 5 main groups (bergamots; lime and German lemon; yuzu and grapefruit; oranges, tangerines, clementines, mandarins, calamansi, and petitgrains; and lemons) and correlate them to the coumarin compositions. | 252558909 |
Bergapten | O=c1ccc2c(OC)c3ccoc3cc2o1 | In this study, we developed and validated a simple and sensitive UPLC-MS/MS method for the detection and quantification of 14 selected oxygen heterocyclic compounds (coumarins and furanocoumarins). Targeted screening using this method was successfully completed for the essential oils of 12 different Citrus species. To our knowledge, this is the most comprehensive investigation of coumarin and furanocoumarin profiles across commercial-scale Citrus oils to date. The lowest amount was detected in calamansi oil. Expressed oil of Italian bergamot showed the highest furanocoumarin content and the highest level of any individual furanocoumarin (bergamottin). Remarkable differences were observed in the coumarin and furanocoumarin levels among oils of different crop varieties and origins within the same species. We found potential correlations between O=c1ccc2c(OC)c3ccoc3cc2o1 and xanthotoxin which matches with known biosynthetic pathways. Patterns in furanocoumarin profiles lined up with known variations among the Citrus ancestral taxa. Using multivariate analysis, we were able to divide the Citrus oils into 5 main groups (bergamots; lime and German lemon; yuzu and grapefruit; oranges, tangerines, clementines, mandarins, calamansi, and petitgrains; and lemons) and correlate them to the coumarin compositions. | 252558909 |
Cofactor | C(N[C@H](C([O-])=O)CCC(=O)[O-])(C1C=CC(=CC=1)N1CC2CNC3N=C(N)NC(C=3N2C1)=O)=O.[Ca+2] | An absence of clear nucleocytoplasmic asymmetry in the trypanosome NPC is remarkable, especially as NPC asymmetry is crucial for driving opisthokont mRNA export [22,89,95]. In particular, the ATP-dependent DEAD box RNA helicase Dbp5 and the RNA export mediator Gle1, with its C(N[C@H](C([O-])=O)CCC(=O)[O-])(C1C=CC(=CC=1)N1CC2CNC3N=C(N)NC(C=3N2C1)=O)=O.[Ca+2] IP 6 (inositol hexakisphosphate), associate with the N-terminal β-propeller of cytoplasmic FG-Nup ScNup159/HsNup214, a member of the ScNup82 complex and remodel messenger ribonucleoproteins (mRNPs) exiting the nucleus [19,20,[23][24][25][96][97][98]. This allows the non-karyopherin RNA export factors (Mex67:Mtr2 in yeast, TAP:p15 in humans) to disengage and recycle back into the nucleus, providing the necessary directionality and energy to RNA export [99][100][101]. As well as lacking a ScNup159/HsNup214 ortholog, orthologs of Gle1 and Dbp5 are absent from affinity-captured complexes and cannot be identified in the trypanosome genome. (See S8 Fig for phylogenetic analysis. Files are viewable using the free \Archaeopteryx\" software.) By contrast | 16132689 |
Inositol hexakisphosphate | P(OC1C(C(OP(O)(O)=O)C(C(OP(=O)(O)O)C1OP(O)(=O)O)OP(O)(O)=O)OP(O)(=O)O)(=O)(O)O | An absence of clear nucleocytoplasmic asymmetry in the trypanosome NPC is remarkable, especially as NPC asymmetry is crucial for driving opisthokont mRNA export [22,89,95]. In particular, the ATP-dependent DEAD box RNA helicase Dbp5 and the RNA export mediator Gle1, with its cofactor IP 6 (P(OC1C(C(OP(O)(O)=O)C(C(OP(=O)(O)O)C1OP(O)(=O)O)OP(O)(O)=O)OP(O)(=O)O)(=O)(O)O), associate with the N-terminal β-propeller of cytoplasmic FG-Nup ScNup159/HsNup214, a member of the ScNup82 complex and remodel messenger ribonucleoproteins (mRNPs) exiting the nucleus [19,20,[23][24][25][96][97][98]. This allows the non-karyopherin RNA export factors (Mex67:Mtr2 in yeast, TAP:p15 in humans) to disengage and recycle back into the nucleus, providing the necessary directionality and energy to RNA export [99][100][101]. As well as lacking a ScNup159/HsNup214 ortholog, orthologs of Gle1 and Dbp5 are absent from affinity-captured complexes and cannot be identified in the trypanosome genome. (See S8 Fig for phylogenetic analysis. Files are viewable using the free \Archaeopteryx\" software.) By contrast | 16132689 |
Phenylalanine | OC([C@H](Cc1ccccc1)N)=O | Besides TbNups, TbMex67/TbMtr2 forms a complex with Ran and other putative Ran binding proteins (RanBP1 and GAP TbTBC-RootA). It is unclear whether TbMex67/Mtr2 can bind Ran directly or is doing so via these other proteins (Fig 6A). If direct, presumably the interaction would be via the NTF2-like domains of Mex67 and Mtr2. NTF2 binds to and imports Ran-GDP into the nucleus [107][108][109][110]. Ran binds NTF2 via a highly conserved OC([C@H](Cc1ccccc1)N)=O (Phe72), called the \switch II\" region | 16132689 |
Inositol hexakisphosphate | C1(OP(O)(O)=O)C(OP(=O)(O)O)C(OP(=O)(O)O)C(C(C1OP(O)(O)=O)OP(O)(O)=O)OP(=O)(O)O | The high level of conservation of inner ring features extends to TbNup65, the ortholog of ScNup53/HsNup35. TbNup65 interacts with the nuclear membrane via an orthodox TM that is conserved between kinetoplastids (S4 Fig) and represents the sole membrane anchor identified in the trypanosome NPC by our methods. That ALPS and TM domains appear functionally interchangeable suggests that the precise mechanism of anchoring the NPC to the nuclear membrane is unimportant, so long as it has some such mechanism (Fig 7A). This idea is supported by the observation that deletion of all TM proteins from A. nidulans NPCs has no deleterious effects on viability (although the putative ALPS-containing proteins are essential in this context) [81]. The absence of an ortholog to the TM protein ScPom152 in trypanosomes is notable, as orthologs are present in other opisthokonts and plants. Pom152 has a cadherin domain, in common with many membrane receptors and proteins that bridge between two membranes [9]. Thus, while this could reflect lineage-specific loss from trypanosomes, a more attractive interpretation is as an example of neofunctionalisation of a membrane protein into a NPC-specific role, postdating speciation between opisthokonts, plants, and trypanosomes. [104] that shows similarity to Rab GTPase Activating Proteins (GAPs) by protein domain prediction. These interactions are suggestive of a role for the Ran gradient in the export of bulk polyA mRNA export. Under low stringency conditions, the interaction between TbMex67 and the TbNPC is clearly observed in a manner reminiscent to that of yeast Mex67 [40]. (B) Models of the trypanosome NTF2, TbMtr2, and the NTF2 domain of TbMex67 were generated using I-TASSER, resulting in C-scores of 1.14, -0.96, and 0.95, respectively. The C-score is used to assess the quality of a model generated by I-TASSER [105]. Its calculation is based on the Z-score of individual threading alignments and the convergence parameters of the I-TASSER assembly simulations. C-scores range between -5 and 2; the closer the score to 2, the higher the confidence in the model generated. The C-scores generated for our models are closer to 2, reflecting high confidence in the models generated. TbNTF2 is capable of binding Ran, based on an accessible potential Ran-binding pocket [106,107], whereas the potential Ran-binding pocket in TbMtr2 and the NTF2 domain of TbMex67 are predicted to be inaccessible, based on structural modeling using I-TASSER. Significantly, this mirrors the situation in yeast and vertebrates, suggesting that Ran binding may not be direct and probably requires the other Ran interacting proteins such as RanBP1 and TbTBC-RootA. Only one copy of the inner ring is illustrated for simplicity. The anchoring mechanism of the TbNPC is provided by a single inner ring Nup (TbNup65) that in yeast (ScNup53/59) interacts with the NE via an ALPS motif. Trypanosomes lack the whole pore membrane ring comprised of Pom152 (GP210 in humans and plants), Pom34, and NDC1 [5,6]. The TbNPC is largely symmetric, with asymmetry provided by its nucleoplasmic interactions through two nuclear basket Nups that are half the size of their opisthokont analogs [35]. Significantly, there are no clear orthologs of Dbp5 and Gle1, coincident with the lack of cytoplasmic or nucleoplasmic biased FG-Nups in trypanosomes. Instead, TbNup76, the candidate ortholog of the cytoplasm-specific Nup82/88 in opisthokonts, localizes to both faces of the NPC. (B) Left, model highlighting the conserved inner ring core (blue) and differences in asymmetry (red) in excavates and opisthokonts as represented by trypanosomes and yeast. Orthologs of cytoplasmic Nups or mRNA remodeling factors are absent from trypanosomes. Right, affinity capture of the conserved nonkaryopherin RNA exporter Mex67 co-isolates Ran, suggesting a putative role for the GTPase Ran in mRNA export in trypanosomes (see Fig 6A). Bulk polyA mRNA export in opisthokonts is driven by ATP through the actions of the ATP-dependent DEAD box helicase DBP5, RNA export factor Gle1, and C1(OP(O)(O)=O)C(OP(=O)(O)O)C(OP(=O)(O)O)C(C(C1OP(O)(O)=O)OP(O)(O)=O)OP(=O)(O)O (IP 6 ) [22]. | 16132689 |
Dimer | CC(C)(C1=CC(=CC(C(C)(C)C)=C1P1C2=CC=CC=C2C(C2=CC=C(C=C2)C2=C(C3=CC=CC=C3)P(C3C2=CC=CC=3)C2C(=CC(=CC=2C(C)(C)C)C(C)(C)C)C(C)(C)C)=C1C1C=CC=CC=1)C(C)(C)C)C | The trypanosome NPC architecture supports our earlier model of NPC evolution, which proposed that the ancestral NPC was an ungated pore, with protocoatomer type subunits stabilizing fenestrations in the protoeukaryotic NE [38]. Conservation of the core scaffold, and the presence of the same folds throughout the scaffold, supports a basic tenet of this model, i.e., that the elaborate architecture of the NPC arose through repeated duplication events from a simple progenitor coating complex. Even the eight-fold symmetry, conserved in trypanosomes [131], suggests a model for a stepwise monomer to CC(C)(C1=CC(=CC(C(C)(C)C)=C1P1C2=CC=CC=C2C(C2=CC=C(C=C2)C2=C(C3=CC=CC=C3)P(C3C2=CC=CC=3)C2C(=CC(=CC=2C(C)(C)C)C(C)(C)C)C(C)(C)C)=C1C1C=CC=CC=1)C(C)(C)C)C to tetramer to octamer transition during evolution. Of significance is that membrane anchoring of protocoatomer systems is promiscuous [11], consistent with divergent NPC membrane tethering described here. Selective gating by FG-Nups was proposed as a more recent acquisition, facilitating more selectivity in import and export [27]. Nevertheless, the high degree of conservation found in the inner ring complex, which contains representatives of all the major elements of the transport machinery (coatomer, karyopherin, FG Nup, membrane association), suggests an intermediate but simpler architecture for a transitional pre-LECA NPC. We propose that, subsequently, a more elaborate architecture evolved, leading to differentiated inner and outer rings and peripheral structures, and providing specific and different functionalities at the nuclear versus cytoplasmic sites. This allowed the development, in particular, of elaborations in mRNP processing and assembly at the NPC's nucleoplasmic face and ATP-dependent export and unloading on the cytoplasmic face. This may also have driven remodeling of FG-Nup positioning, with the trypanosome symmetric arrangement perhaps reflecting that in the LECA NPC, and being consistent with the trypanosomatid lineage as one of the earliest to differentiate following the eukaryogenesis event. | 16132689 |
Tetramer | P1(N=[P@@](Cl)(N=[P@](Nc2cc3c(cc2)OCCO3)(Cl)N=P(N=1)(Cl)Cl)Nc1cc2c(cc1)OCCO2)(Cl)Cl | The trypanosome NPC architecture supports our earlier model of NPC evolution, which proposed that the ancestral NPC was an ungated pore, with protocoatomer type subunits stabilizing fenestrations in the protoeukaryotic NE [38]. Conservation of the core scaffold, and the presence of the same folds throughout the scaffold, supports a basic tenet of this model, i.e., that the elaborate architecture of the NPC arose through repeated duplication events from a simple progenitor coating complex. Even the eight-fold symmetry, conserved in trypanosomes [131], suggests a model for a stepwise monomer to dimer to P1(N=[P@@](Cl)(N=[P@](Nc2cc3c(cc2)OCCO3)(Cl)N=P(N=1)(Cl)Cl)Nc1cc2c(cc1)OCCO2)(Cl)Cl to octamer transition during evolution. Of significance is that membrane anchoring of protocoatomer systems is promiscuous [11], consistent with divergent NPC membrane tethering described here. Selective gating by FG-Nups was proposed as a more recent acquisition, facilitating more selectivity in import and export [27]. Nevertheless, the high degree of conservation found in the inner ring complex, which contains representatives of all the major elements of the transport machinery (coatomer, karyopherin, FG Nup, membrane association), suggests an intermediate but simpler architecture for a transitional pre-LECA NPC. We propose that, subsequently, a more elaborate architecture evolved, leading to differentiated inner and outer rings and peripheral structures, and providing specific and different functionalities at the nuclear versus cytoplasmic sites. This allowed the development, in particular, of elaborations in mRNP processing and assembly at the NPC's nucleoplasmic face and ATP-dependent export and unloading on the cytoplasmic face. This may also have driven remodeling of FG-Nup positioning, with the trypanosome symmetric arrangement perhaps reflecting that in the LECA NPC, and being consistent with the trypanosomatid lineage as one of the earliest to differentiate following the eukaryogenesis event. | 16132689 |
Paraformaldehyde | C=O | GFP-tagged cell lines were harvested and fixed for 10 mins in a final concentration of 2% C=O. Fixed cells were then washed in 1xPBS and visualized as previously described [27]. | 16132689 |
Dithiothreitol | O[C@H](CS)[C@@H](CS)O | Trypanosomes were grown to a density of between 2.5 x 10 7 cells per ml. Parasites were harvested by centrifugation, washed in 1xPBS with protease inhibitors and 10mM O[C@H](CS)[C@@H](CS)O, and flash frozen in liquid nitrogen to preserve protein:protein interactions as close as they were at time of freezing as possible. Cells were cryomilled into a fine grindate in a planetary ball mill (Retsch). For a very detailed protocol, refer to Obado et al., 2015 (in press), Methods in Molecular Biology, or the National Center for Dynamic Interactome Research website (www.NCDIR. org/protocols). Cryomilled cellular materials were resuspended in various extraction buffers (S1 Table) containing a protease inhibitor cocktail without EDTA (Roche), sonicated on ice with a microtip sonicator (Misonix Ultrasonic Processor XL) at Setting 4 (~20W output) for 2 x 1 second to break apart aggregates that may be invisible to the eye, and clarified by centrifugation (20,000 x g) for 10 min at 4°C (Obado et al., 2015 (in press), Methods in Molecular Biology, or www.NCDIR.org/protocols) [41]. Clarified lysates were incubated with magnetic beads conjugated with polyclonal anti-GFP llama antibodies on a rotator for 1 hr at 4°C. The magnetic beads were harvested by magnetization (Dynal) and washed three times with extraction buffer prior to elution with 2% SDS/40 mM Tris pH 8.0. The eluate was reduced in 50 mM DTT and alkylated with 100 mM iodoacetamide prior to downstream analysis (SDS-PAGE followed by protein identification using MS-electrospray ionization (ESI) or MALDI-TOF). Eluates were fractionated on precast Novex 4-12% Bis Tris gels (Life Technology), stained using colloidal Coomassie (GelCode Blue-Thermo) and analyzed by MS [27]. | 16132689 |
Iodoacetamide | NC(CI)=O | Trypanosomes were grown to a density of between 2.5 x 10 7 cells per ml. Parasites were harvested by centrifugation, washed in 1xPBS with protease inhibitors and 10mM dithiothreitol, and flash frozen in liquid nitrogen to preserve protein:protein interactions as close as they were at time of freezing as possible. Cells were cryomilled into a fine grindate in a planetary ball mill (Retsch). For a very detailed protocol, refer to Obado et al., 2015 (in press), Methods in Molecular Biology, or the National Center for Dynamic Interactome Research website (www.NCDIR. org/protocols). Cryomilled cellular materials were resuspended in various extraction buffers (S1 Table) containing a protease inhibitor cocktail without EDTA (Roche), sonicated on ice with a microtip sonicator (Misonix Ultrasonic Processor XL) at Setting 4 (~20W output) for 2 x 1 second to break apart aggregates that may be invisible to the eye, and clarified by centrifugation (20,000 x g) for 10 min at 4°C (Obado et al., 2015 (in press), Methods in Molecular Biology, or www.NCDIR.org/protocols) [41]. Clarified lysates were incubated with magnetic beads conjugated with polyclonal anti-GFP llama antibodies on a rotator for 1 hr at 4°C. The magnetic beads were harvested by magnetization (Dynal) and washed three times with extraction buffer prior to elution with 2% SDS/40 mM Tris pH 8.0. The eluate was reduced in 50 mM DTT and alkylated with 100 mM NC(CI)=O prior to downstream analysis (SDS-PAGE followed by protein identification using MS-electrospray ionization (ESI) or MALDI-TOF). Eluates were fractionated on precast Novex 4-12% Bis Tris gels (Life Technology), stained using colloidal Coomassie (GelCode Blue-Thermo) and analyzed by MS [27]. | 16132689 |
Novex | c1(O)c(Sc2cc(Cl)ccc2O)cc(Cl)cc1 | Trypanosomes were grown to a density of between 2.5 x 10 7 cells per ml. Parasites were harvested by centrifugation, washed in 1xPBS with protease inhibitors and 10mM dithiothreitol, and flash frozen in liquid nitrogen to preserve protein:protein interactions as close as they were at time of freezing as possible. Cells were cryomilled into a fine grindate in a planetary ball mill (Retsch). For a very detailed protocol, refer to Obado et al., 2015 (in press), Methods in Molecular Biology, or the National Center for Dynamic Interactome Research website (www.NCDIR. org/protocols). Cryomilled cellular materials were resuspended in various extraction buffers (S1 Table) containing a protease inhibitor cocktail without EDTA (Roche), sonicated on ice with a microtip sonicator (Misonix Ultrasonic Processor XL) at Setting 4 (~20W output) for 2 x 1 second to break apart aggregates that may be invisible to the eye, and clarified by centrifugation (20,000 x g) for 10 min at 4°C (Obado et al., 2015 (in press), Methods in Molecular Biology, or www.NCDIR.org/protocols) [41]. Clarified lysates were incubated with magnetic beads conjugated with polyclonal anti-GFP llama antibodies on a rotator for 1 hr at 4°C. The magnetic beads were harvested by magnetization (Dynal) and washed three times with extraction buffer prior to elution with 2% SDS/40 mM Tris pH 8.0. The eluate was reduced in 50 mM DTT and alkylated with 100 mM iodoacetamide prior to downstream analysis (SDS-PAGE followed by protein identification using MS-electrospray ionization (ESI) or MALDI-TOF). Eluates were fractionated on precast c1(O)c(Sc2cc(Cl)ccc2O)cc(Cl)cc1 4-12% Bis Tris gels (Life Technology), stained using colloidal Coomassie (GelCode Blue-Thermo) and analyzed by MS [27]. | 16132689 |
Ammonium | [NH4+] | Briefly, protein bands were excised from acrylamide gels and destained using 50% acetonitrile, 40% water, and 10% [NH4+] bicarbonate (v/v/w). Gel pieces were dried and resuspended in trypsin digestion buffer; 50 mM [NH4+] bicarbonate, pH 7.5, 10% acetonitrile, and 0.1-2 ug sequence-grade trypsin, depending on protein band intensity. Digestion was carried out at 37°C for 6 h prior to peptide extraction using C18 beads (POROS) in 2% TFA (trifluoroacetic acid) and 5% formamide. Extracted peptides were washed in 0.1% acetic acid (ESI) or 0.1% TFA (MALDI) and analyzed on a LTQ Velos (ESI) (Thermo) or pROTOF (MALDI-TOF) (PerkinElmer). | 16132689 |
Bicarbonate | C(O)([O-])=O | Briefly, protein bands were excised from acrylamide gels and destained using 50% acetonitrile, 40% water, and 10% ammonium C(O)([O-])=O (v/v/w). Gel pieces were dried and resuspended in trypsin digestion buffer; 50 mM ammonium C(O)([O-])=O, pH 7.5, 10% acetonitrile, and 0.1-2 ug sequence-grade trypsin, depending on protein band intensity. Digestion was carried out at 37°C for 6 h prior to peptide extraction using C18 beads (POROS) in 2% TFA (trifluoroacetic acid) and 5% formamide. Extracted peptides were washed in 0.1% acetic acid (ESI) or 0.1% TFA (MALDI) and analyzed on a LTQ Velos (ESI) (Thermo) or pROTOF (MALDI-TOF) (PerkinElmer). | 16132689 |
Formamide | O=CN | Briefly, protein bands were excised from acrylamide gels and destained using 50% acetonitrile, 40% water, and 10% ammonium bicarbonate (v/v/w). Gel pieces were dried and resuspended in trypsin digestion buffer; 50 mM ammonium bicarbonate, pH 7.5, 10% acetonitrile, and 0.1-2 ug sequence-grade trypsin, depending on protein band intensity. Digestion was carried out at 37°C for 6 h prior to peptide extraction using C18 beads (POROS) in 2% TFA (trifluoroacetic acid) and 5% O=CN. Extracted peptides were washed in 0.1% acetic acid (ESI) or 0.1% TFA (MALDI) and analyzed on a LTQ Velos (ESI) (Thermo) or pROTOF (MALDI-TOF) (PerkinElmer). | 16132689 |
Acetone | C(=O)(C)C | Trypanosomes were cryoprotected with 20% bovine serum albumin (BSA) and applied to a high pressure freezing procedure (EMPACT2, Leica Microsystem System, Wetzlar, Germany). Cells were transferred to a freeze substitution device (EM AFS2, Leica Microsystem System, Wetzlar, Germany), incubated with 0.2% Uranyl acetate in 95% C(=O)(C)C at -90 C°, and embed in Lowicryl HM20 at -35 C°. Ultrathin sections were cut and post-embedding immunostaining was applied. Briefly, sections were blocked with 2% BSA plus 0.1% saponin in Tris buffered saline (TBS; 20 mM Tris-Cl, pH 7.5, 150 mM NaCl) for 30 min. Sections were then incubated in fresh blocking solution containing polyclonal rabbit anti-GFP antibodies (1:150) overnight at 4°C, and washed with TBS the next day. The EM sections were then incubated overnight with secondary goat anti-rabbit antibodies conjugated with 12 nm colloidal gold (1:20) in 0.2% BSA plus 0.1% saponin in TBS and then washed in TBS buffer. An additional wash step using 1 x PBS was performed prior to fixation for 5 min with 2.5% glutaraldehyde. Post fixed grids were washed with water and uranyl acetate (1%), and lead citrate (1%) was applied. The sections were examined in the electron microscope (100CX JEOL, Tokyo, Japan) with the digital imaging system (XR41-C, AMT Imaging, Woburn, Massachusetts). Control experiments were done by following the same procedure, except for the omission of primary antibody and applying just the blocking solution instead. | 16132689 |
Glutaraldehyde | C(CC=O)CC=O | Trypanosomes were cryoprotected with 20% bovine serum albumin (BSA) and applied to a high pressure freezing procedure (EMPACT2, Leica Microsystem System, Wetzlar, Germany). Cells were transferred to a freeze substitution device (EM AFS2, Leica Microsystem System, Wetzlar, Germany), incubated with 0.2% Uranyl acetate in 95% acetone at -90 C°, and embed in Lowicryl HM20 at -35 C°. Ultrathin sections were cut and post-embedding immunostaining was applied. Briefly, sections were blocked with 2% BSA plus 0.1% saponin in Tris buffered saline (TBS; 20 mM Tris-Cl, pH 7.5, 150 mM NaCl) for 30 min. Sections were then incubated in fresh blocking solution containing polyclonal rabbit anti-GFP antibodies (1:150) overnight at 4°C, and washed with TBS the next day. The EM sections were then incubated overnight with secondary goat anti-rabbit antibodies conjugated with 12 nm colloidal gold (1:20) in 0.2% BSA plus 0.1% saponin in TBS and then washed in TBS buffer. An additional wash step using 1 x PBS was performed prior to fixation for 5 min with 2.5% C(CC=O)CC=O. Post fixed grids were washed with water and uranyl acetate (1%), and lead citrate (1%) was applied. The sections were examined in the electron microscope (100CX JEOL, Tokyo, Japan) with the digital imaging system (XR41-C, AMT Imaging, Woburn, Massachusetts). Control experiments were done by following the same procedure, except for the omission of primary antibody and applying just the blocking solution instead. | 16132689 |
Lead citrate | [O-]C(=O)CC(CC([O-])=O)(C(=O)[O-])O.C(CC(O)(CC([O-])=O)C(=O)[O-])(=O)[O-].[Pb+2].[Pb+2].[Pb+2] | Trypanosomes were cryoprotected with 20% bovine serum albumin (BSA) and applied to a high pressure freezing procedure (EMPACT2, Leica Microsystem System, Wetzlar, Germany). Cells were transferred to a freeze substitution device (EM AFS2, Leica Microsystem System, Wetzlar, Germany), incubated with 0.2% Uranyl acetate in 95% acetone at -90 C°, and embed in Lowicryl HM20 at -35 C°. Ultrathin sections were cut and post-embedding immunostaining was applied. Briefly, sections were blocked with 2% BSA plus 0.1% saponin in Tris buffered saline (TBS; 20 mM Tris-Cl, pH 7.5, 150 mM NaCl) for 30 min. Sections were then incubated in fresh blocking solution containing polyclonal rabbit anti-GFP antibodies (1:150) overnight at 4°C, and washed with TBS the next day. The EM sections were then incubated overnight with secondary goat anti-rabbit antibodies conjugated with 12 nm colloidal gold (1:20) in 0.2% BSA plus 0.1% saponin in TBS and then washed in TBS buffer. An additional wash step using 1 x PBS was performed prior to fixation for 5 min with 2.5% glutaraldehyde. Post fixed grids were washed with water and uranyl acetate (1%), and [O-]C(=O)CC(CC([O-])=O)(C(=O)[O-])O.C(CC(O)(CC([O-])=O)C(=O)[O-])(=O)[O-].[Pb+2].[Pb+2].[Pb+2] (1%) was applied. The sections were examined in the electron microscope (100CX JEOL, Tokyo, Japan) with the digital imaging system (XR41-C, AMT Imaging, Woburn, Massachusetts). Control experiments were done by following the same procedure, except for the omission of primary antibody and applying just the blocking solution instead. | 16132689 |
Citrate | C(C(O)(C([O-])=O)CC(=O)[O-])C([O-])=O | Trypanosomes were cryoprotected with 20% bovine serum albumin (BSA) and applied to a high pressure freezing procedure (EMPACT2, Leica Microsystem System, Wetzlar, Germany). Cells were transferred to a freeze substitution device (EM AFS2, Leica Microsystem System, Wetzlar, Germany), incubated with 0.2% Uranyl acetate in 95% acetone at -90 C°, and embed in Lowicryl HM20 at -35 C°. Ultrathin sections were cut and post-embedding immunostaining was applied. Briefly, sections were blocked with 2% BSA plus 0.1% saponin in Tris buffered saline (TBS; 20 mM Tris-Cl, pH 7.5, 150 mM NaCl) for 30 min. Sections were then incubated in fresh blocking solution containing polyclonal rabbit anti-GFP antibodies (1:150) overnight at 4°C, and washed with TBS the next day. The EM sections were then incubated overnight with secondary goat anti-rabbit antibodies conjugated with 12 nm colloidal gold (1:20) in 0.2% BSA plus 0.1% saponin in TBS and then washed in TBS buffer. An additional wash step using 1 x PBS was performed prior to fixation for 5 min with 2.5% glutaraldehyde. Post fixed grids were washed with water and uranyl acetate (1%), and lead C(C(O)(C([O-])=O)CC(=O)[O-])C([O-])=O (1%) was applied. The sections were examined in the electron microscope (100CX JEOL, Tokyo, Japan) with the digital imaging system (XR41-C, AMT Imaging, Woburn, Massachusetts). Control experiments were done by following the same procedure, except for the omission of primary antibody and applying just the blocking solution instead. | 16132689 |
Flexor | Cc1ccccc1C(c1ccccc1)OCCN(C)C.C(C(O)=O)C(CC(O)=O)(C(=O)O)O | Maximum extension angles were significantly lower in all joints, with respect to controls (p < 0.001). Deficit of finger extension has been demonstrated to be the results of two concurrent causes: mechanical restraint to extension and altered neurophysiological control mechanisms. A number of studies have documented changes in the mechanical properties of upper limb muscles. In particular, atrophy of extensors [32] and contractures of Cc1ccccc1C(c1ccccc1)OCCN(C)C.C(C(O)=O)C(CC(O)=O)(C(=O)O)Os caused by shortening of muscle fibres and increased passive stiffness of muscular tissue [23] have been demonstrated to contribute to limit fingers extension. However, deficit in hand opening has been documented also in stroke subjects who didn't present with increased passive resistance [33], suggesting that anomalies in neurological control play a major role in reducing finger joints motion. Three main neuromotor causes have been demonstrated to interfere with hand opening. The first alteration is Cc1ccccc1C(c1ccccc1)OCCN(C)C.C(C(O)=O)C(CC(O)=O)(C(=O)O)Os spasticity, an involuntary velocitydependent contraction of Cc1ccccc1C(c1ccccc1)OCCN(C)C.C(C(O)=O)C(CC(O)=O)(C(=O)O)O muscles during finger extension due to an exaggerated stretch reflex activity [33,34]. The other two aspects are excessive co-contraction of Cc1ccccc1C(c1ccccc1)OCCN(C)C.C(C(O)=O)C(CC(O)=O)(C(=O)O)Os and extensors [5,35] and weakness of extensor muscles, presumably caused by a reduction in the activation of spinal segmental neurons [36]. | 17617875 |
Flexor | N(C)(C)CCOC(c1ccccc1)c1ccccc1C.O=C(O)CC(C(=O)O)(CC(=O)O)O | Inspection of each stroke subject, revealed the existence of four different behaviours of the hemiparetic hand during opening. Of fourteen hands analysed, one was almost unaltered (type 0 hand), seven had uniform involvement of all long fingers (type I, type II and type III hands), while six showed differential impairment among digits (type MIX hands). Type I fingers showed a nearly normal motion of IPJ and a reduced extension of MCPJ, associated with a reverse inter-joint coordination sequence (i.e. distal-to-proximal). As reported by Kamper et al [35], the weakness of extrinsic extensors (i.e. extensor digitorum communis) and the exaggerated cocontraction of extrinsic N(C)(C)CCOC(c1ccccc1)c1ccccc1C.O=C(O)CC(C(=O)O)(CC(=O)O)Os (i.e. N(C)(C)CCOC(c1ccccc1)c1ccccc1C.O=C(O)CC(C(=O)O)(CC(=O)O)O digitorum profundus) could justify the reduced motion of MCPJ, while a good activation of intrinsic muscles (interossei and lumbricals) could explain the physiological extension of IPJ. The reversed distal-to-proximal synergy has been demonstrated to be partly due to a delayed motion of MCPJ (see Figure 10c) possibly explained by an abnormally high brake action of extrinsic N(C)(C)CCOC(c1ccccc1)c1ccccc1C.O=C(O)CC(C(=O)O)(CC(=O)O)Os [30], and partly caused by a significantly slower movement of MCPJ (see Figure 10d) possibly due to slow and weak activation of extensor digitorum communis. Contrarily to type I, type II digits revealed impairment of IPJ extension only, with a significantly high delay between IPJ and MCPJ in long fingers. This pattern of movement appeared similar to the task of voluntary curling the fingers while extending MCPJ, described by Long & Brown [30] in healthy controls. During this task, the authors reported the co-activation of extensor digitorum communis and N(C)(C)CCOC(c1ccccc1)c1ccccc1C.O=C(O)CC(C(=O)O)(CC(=O)O)O digitorum profundus, with silent activity of lumbricals and interossei (prime extensors of IPJ). From this comparison, it can be speculated that type II fingers could show a physiological activation of extensor digitorum communis, an abnormally high co-activation of extrinsic N(C)(C)CCOC(c1ccccc1)c1ccccc1C.O=C(O)CC(C(=O)O)(CC(=O)O)Os and a severe weakness of intrinsic muscles (lumbricals and interossei), which in turn, would explain the unimpaired movement of MCPJ and the reduced extension of IPJ. The high IPJ-MCPJ delay has been demonstrated to be due, in 30% of the cases, to a segmented movement in which IPJ start moving after MCPJ has already reached full extension (see Figure 10e) and, in 70% of the cases, to an abnormal slowness of IPJ in completing the movement (see Figure 10f). In the first case the high value of parameter Δ could be caused by a delayed but fast activation of lumbricals which generates a stretch reflex on IPJ N(C)(C)CCOC(c1ccccc1)c1ccccc1C.O=C(O)CC(C(=O)O)(CC(=O)O)Os, while, in the second case it could be explained mainly by lumbrical weakness and slow prolonged activation, rather than to a delayed reclutation of muscle fibers. Finally, the most impaired hands (type III), which revealed reduction of both MCPJ and IPJ extension, possibly show all the muscle activity anomalies described for type I and type II hands. | 17617875 |
Lumbrical | C1NCCNC1 | Inspection of each stroke subject, revealed the existence of four different behaviours of the hemiparetic hand during opening. Of fourteen hands analysed, one was almost unaltered (type 0 hand), seven had uniform involvement of all long fingers (type I, type II and type III hands), while six showed differential impairment among digits (type MIX hands). Type I fingers showed a nearly normal motion of IPJ and a reduced extension of MCPJ, associated with a reverse inter-joint coordination sequence (i.e. distal-to-proximal). As reported by Kamper et al [35], the weakness of extrinsic extensors (i.e. extensor digitorum communis) and the exaggerated cocontraction of extrinsic flexors (i.e. flexor digitorum profundus) could justify the reduced motion of MCPJ, while a good activation of intrinsic muscles (interossei and C1NCCNC1s) could explain the physiological extension of IPJ. The reversed distal-to-proximal synergy has been demonstrated to be partly due to a delayed motion of MCPJ (see Figure 10c) possibly explained by an abnormally high brake action of extrinsic flexors [30], and partly caused by a significantly slower movement of MCPJ (see Figure 10d) possibly due to slow and weak activation of extensor digitorum communis. Contrarily to type I, type II digits revealed impairment of IPJ extension only, with a significantly high delay between IPJ and MCPJ in long fingers. This pattern of movement appeared similar to the task of voluntary curling the fingers while extending MCPJ, described by Long & Brown [30] in healthy controls. During this task, the authors reported the co-activation of extensor digitorum communis and flexor digitorum profundus, with silent activity of C1NCCNC1s and interossei (prime extensors of IPJ). From this comparison, it can be speculated that type II fingers could show a physiological activation of extensor digitorum communis, an abnormally high co-activation of extrinsic flexors and a severe weakness of intrinsic muscles (C1NCCNC1s and interossei), which in turn, would explain the unimpaired movement of MCPJ and the reduced extension of IPJ. The high IPJ-MCPJ delay has been demonstrated to be due, in 30% of the cases, to a segmented movement in which IPJ start moving after MCPJ has already reached full extension (see Figure 10e) and, in 70% of the cases, to an abnormal slowness of IPJ in completing the movement (see Figure 10f). In the first case the high value of parameter Δ could be caused by a delayed but fast activation of C1NCCNC1s which generates a stretch reflex on IPJ flexors, while, in the second case it could be explained mainly by C1NCCNC1 weakness and slow prolonged activation, rather than to a delayed reclutation of muscle fibers. Finally, the most impaired hands (type III), which revealed reduction of both MCPJ and IPJ extension, possibly show all the muscle activity anomalies described for type I and type II hands. | 17617875 |
Flexor | C1(=CC=CC=C1)C(C1C(=CC=CC=1)C)OCCN(C)C.O=C(CC(C(O)=O)(CC(O)=O)O)O | In three cases, subjects attempts to open their hand resulted in an inappropriate flexion of one or two IPJs of the hand, as found also by Kamper et al [35]. Again, the origin of this anomalous behaviour could be ascribed to the exaggerated co-activation of C1(=CC=CC=C1)C(C1C(=CC=CC=1)C)OCCN(C)C.O=C(CC(C(O)=O)(CC(O)=O)O)O muscles, possibly due to the loss of descending inputs involved in reciprocal inhibition of C1(=CC=CC=C1)C(C1C(=CC=CC=1)C)OCCN(C)C.O=C(CC(C(O)=O)(CC(O)=O)O)O muscles [37] and/or to a preferential activation of cortical neurons responsible for co-contraction of antagonists muscles [35]. | 17617875 |
Agarose | O[C@H]1[C@H]2[C@H](O[C@H]3[C@H](O)[C@H](O[C@H]4[C@H]([C@@H]5OC[C@H](O4)[C@H]5O[C@H]4[C@H](O)[C@@H](O)[C@@H](O)[C@H](O4)CO)O)[C@H]([C@@H](CO)O3)O)[C@@H](O[C@H]1O)CO2 | To confirm the specific amplification of primers, PCR assay using two outer primers including Eb-B3 and Eb-F3, which amplifies ~274-bp fragment of the target gene, was employed.The PCR amplification was performed in a 15 μL volume consisted of 7.5 μL of 2× Taq red master mix (Ampliqon, Odense, Denmark), 10 ρM of each forward (Eb-F3) and reverse (Eb-B3) primers, and 2 μL of DNA templet.The cycling conditions were: an initial denaturation step of 94 • C for 4 min, followed by 40 cycles of 94 controls, respectively.To identify the optimal temperature and time for LAMP amplification, the reactions were conducted in 60-65 • C. The optimal time was evaluated every 15 min from 30 to 120 min.Specific melting curve indicated specific target amplification.The LAMP products were electrophoresed on a 1.2% O[C@H]1[C@H]2[C@H](O[C@H]3[C@H](O)[C@H](O[C@H]4[C@H]([C@@H]5OC[C@H](O4)[C@H]5O[C@H]4[C@H](O)[C@@H](O)[C@@H](O)[C@H](O4)CO)O)[C@H]([C@@H](CO)O3)O)[C@@H](O[C@H]1O)CO2 gel and visualized using ethidium bromide to confirm LAMP reaction. | 268398272 |
Ethidium | c1cccc(c1)-c1[n+](c2cc(ccc2c2ccc(cc12)N)N)CC | To confirm the specific amplification of primers, PCR assay using two outer primers including Eb-B3 and Eb-F3, which amplifies ~274-bp fragment of the target gene, was employed.The PCR amplification was performed in a 15 μL volume consisted of 7.5 μL of 2× Taq red master mix (Ampliqon, Odense, Denmark), 10 ρM of each forward (Eb-F3) and reverse (Eb-B3) primers, and 2 μL of DNA templet.The cycling conditions were: an initial denaturation step of 94 • C for 4 min, followed by 40 cycles of 94 controls, respectively.To identify the optimal temperature and time for LAMP amplification, the reactions were conducted in 60-65 • C. The optimal time was evaluated every 15 min from 30 to 120 min.Specific melting curve indicated specific target amplification.The LAMP products were electrophoresed on a 1.2% agarose gel and visualized using c1cccc(c1)-c1[n+](c2cc(ccc2c2ccc(cc12)N)N)CC bromide to confirm LAMP reaction. | 268398272 |
Bromide | [Br-] | To confirm the specific amplification of primers, PCR assay using two outer primers including Eb-B3 and Eb-F3, which amplifies ~274-bp fragment of the target gene, was employed.The PCR amplification was performed in a 15 μL volume consisted of 7.5 μL of 2× Taq red master mix (Ampliqon, Odense, Denmark), 10 ρM of each forward (Eb-F3) and reverse (Eb-B3) primers, and 2 μL of DNA templet.The cycling conditions were: an initial denaturation step of 94 • C for 4 min, followed by 40 cycles of 94 controls, respectively.To identify the optimal temperature and time for LAMP amplification, the reactions were conducted in 60-65 • C. The optimal time was evaluated every 15 min from 30 to 120 min.Specific melting curve indicated specific target amplification.The LAMP products were electrophoresed on a 1.2% agarose gel and visualized using ethidium [Br-] to confirm LAMP reaction. | 268398272 |
Agarose | O1[C@@H]2[C@H]([C@H]([C@@H]([C@@H]1O[C@@H]1[C@@H](O)[C@@H](CO)O[C@@H](O[C@H]3[C@H]4OC[C@@H]3O[C@@H](O)[C@H]4O)[C@@H]1O)O)OC2)O[C@H]1[C@H](O)[C@H]([C@@H](O)[C@H](O1)CO)O | The result of in silico analyses and conventional PCR with Eb-F3 and Eb-B3 primers showed a ~ 274-bp band in electrophoresis, which verifies the specific binding of primers to the target gene.To determine the results of LAMP assay, amplification plots generated by the real-time PCR instrument were considered.The results showed that the 63 • C for 60 min is the best condition for LAMP assay (Fig. 1A, B, and C, Suppl Fig. 1).The validity of amplification was evaluated using melting curve analysis.The average time and melting curve values were 32.3 ± 8.8 min (17.5-46) and 89.5 • C ± 0.8 (88.5-91.2),respectively.Accordingly, from 30 nested-PCR positive samples, 25 (83.3%) were positive by the LAMP method (Table 2).All LAMP products were electrophoresed on O1[C@@H]2[C@H]([C@H]([C@@H]([C@@H]1O[C@@H]1[C@@H](O)[C@@H](CO)O[C@@H](O[C@H]3[C@H]4OC[C@@H]3O[C@@H](O)[C@H]4O)[C@@H]1O)O)OC2)O[C@H]1[C@H](O)[C@H]([C@@H](O)[C@H](O1)CO)O gel that the results confirmed the results of LAMP assay (Fig. 2). | 268398272 |
Distilled water | O | The LAMP reaction was performed in a Rotor-Gene Q(QIAGEN, Germany) real-time instrument by Eva Green fluorescence dye for E. bieneusi-positive samples.The amplification was carried out in a 25 μL PCR reaction mix containing: 40 pmol of each Eb-FIP and Eb-BIP primer, 5 pmol of each Eb-F3 and Eb-B3 primer, 8 U of Bst 2 DNA polymerase (New England Biolabs, USA), 1.4 mmol/L of deoxynucleoside triphosphates (dNTP), 2.5 μL 10× isothermal amplification buffer (New England Biolabs, USA), 6 mmol/L of MgSO 4 , and 2 μL of extracted DNA.O and a previously confirmed DNA for E. bieneusi were employed as negative and positive | 268398272 |
Raw material | O(C)C(=O)[C@H](N)CC1=CN=CN1.Cl | Firstly, orders are defined in the system. In the second step, all machinery (sources) and their capacities within the production environment are defined. In the third step, the routes between the machines are defined. In the fourth, the rules and matrixes required for integration of assets (O(C)C(=O)[C@H](N)CC1=CN=CN1.Cl, semi products) during the production process, were established. In the last step, the animation of overall system is prepared and synchronized with the model. Production line is left as it is after finishing the 8 h of daily work and continues from there in the next working day. So reoccurring of steady state is beside the point as long as orders of that product are not completed. In other words production line is continuously produce until the orders of a product is finished. Therefore type of this system is non-terminating. | 1805135 |
Dimer | c1(C(C)(C)C)cc(C(C)(C)C)c(-p2c(-c3ccccc3)c(c3ccccc23)-c2ccc(cc2)-c2c(p(-c3c(cc(cc3C(C)(C)C)C(C)(C)C)C(C)(C)C)c3ccccc32)-c2ccccc2)c(C(C)(C)C)c1 | E. coli contains five distinct Pols, which are named Pols I-V. Pols II, IV and V act in TLS [1,3], while Pol I functions in DNA repair and Okazaki fragment maturation [24]. The 20-subunit Pol III holoenzyme serves as the bacterial replicase, and is composed of 2 homoc1(C(C)(C)C)cc(C(C)(C)C)c(-p2c(-c3ccccc3)c(c3ccccc23)-c2ccc(cc2)-c2c(p(-c3c(cc(cc3C(C)(C)C)C(C)(C)C)C(C)(C)C)c3ccccc32)-c2ccccc2)c(C(C)(C)C)c1ic β sliding clamps and 3 core complexes (Pol IIIαεθ), which are tethered together via interaction of Pol IIIα with the heptameric DnaX ATPase (τ 3 δδ'ψχ) that acts to load the β clamp onto primed DNA [25,26]. The Pol IIIαεθ core complex performs DNA synthesis functions. Within this complex, Pol IIIα catalyzes DNA polymerization, Pol IIIε functions in proofreading and Pol IIIθ modestly stimulates Pol IIIε proofreading activity [25,26]. With the possible exception of Pol I, each of the 5 E. coli Pols contains either a pentameric (QLxLF) or hexameric (QLxLxL) clamp-binding motif (CBM) that is required for biological activity [27][28][29][30][31]. The CBM interacts with a hydrophobic cleft located near the C-terminus of each β clamp protomer. The different Pols also contact non-cleft surfaces of the β clamp, and these interactions likewise contribute to Pol function and/or Pol switching [17,[32][33][34][35][36][37]. To date, structural information regarding these noncleft contacts is restricted to Pol IV. In the co-crystal structure of the complex consisting of the Pol IV little finger domain (Pol IV LF ; residues 243-351) bound to the β clamp, residues 303 VWP 305 of Pol IV interacted with positions E93 and L98 on the rim of the β clamp, while the C-terminal hexameric CBM ( 346 QLVLGL 351 ) of Pol IV extended over the β clamp c1(C(C)(C)C)cc(C(C)(C)C)c(-p2c(-c3ccccc3)c(c3ccccc23)-c2ccc(cc2)-c2c(p(-c3c(cc(cc3C(C)(C)C)C(C)(C)C)C(C)(C)C)c3ccccc32)-c2ccccc2)c(C(C)(C)C)c1 interface to interact with the β clamp cleft of the adjacent β clamp protomer [32]. Using a primer extension assay, we previously demonstrated that while only the Pol IV-β clamp cleft interaction was required for processive replication, both the β clamp rim and cleft interactions contributed to Pol III-Pol IV switching in vitro [12,17,38]. In contrast to our findings, Gabbai and colleagues, utilizing a different assay that may more accurately represent the structure and composition of the replisome, concluded that the Pol IV-β clamp rim contact stimulated but was not required for a Pol III-Pol IV switch in vitro [18]. In light of this finding, they suggested direct competition between Pol III and Pol IV for the β clamp cleft as an alternative mechanism for their switching. In addition to switching, Pol IV can be recruited directly to single strand (ss) DNA gaps generated by Pol III skipping over lesions in the template strand to continue replication downstream of the block [1,39]. In summary, recruitment of TLS Pols to lesions is suggested to occur by two different mechanisms: (i) β clamp may recruit TLS Pols post-replicatively to lesions within ssDNA DNA gaps generated by Pol III skipping, or (ii) TLS Pols may be recruited to the replication fork and access lesions after undergoing a switch with Pol III. However, the extent to which these proposed mechanisms are utilized in vivo has not yet been determined. Furthermore, the biological relevance of the Pol IV-β clamp rim interaction to the TLS function of Pol IV is also unknown. | 7464696 |
Nitrofurazone | O=[N+]([O-])C1=CC=C(O1)/C=N/NC(=O)N | E. coli Pol IV catalyzes accurate bypass of N 2 -dG lesions induced by O=[N+]([O-])C1=CC=C(O1)/C=N/NC(=O)N (NFZ), as well as alkylated adducts such as N 3 -methyladenine (N 3 -mdA) caused by methyl methanesulfonate (MMS). As a result, E. coli strains lacking Pol IV function (i.e., ΔdinB) display sensitivity to these agents [17,[40][41][42][43]. In contrast to this protective role, overproduction of Pol IV is lethal to E. coli [34,44,45]; similarly, aberrant expression of Pol κ, the eukaryotic ortholog of dinB, promotes genome instability in human cells [46]. Lethality in aerobically cultured E. coli cells was suggested to result from toxic levels of double strand (ds) DNA breaks resulting from efforts to repair closely spaced 8-oxo-7,8-deoxyguanosine (8-oxo-dG) adducts incorporated during replication of undamaged DNA by Pol IV [47]. However, sensitivity of a dnaN159 strain to~4-fold higher than SOS-induced levels of Pol IV [34,44], and of a dnaN + E. coli strain to significantly higher than SOS-induced levels (i.e.,~70-fold; [45]), were both independent of Pol IV catalytic activity, suggesting that at least in these cases, lethality relied on one or more alternative mechanisms. The dnaN159 allele encodes a mutant form of the β sliding clamp that is deficient in regulation of proper access of the different E. coli Pols to DNA [34,35,38,44,48,49]. Thus, sensitivity of the dnaN159 strain to elevated levels of Pol IV was suggested to result from its enhanced ability to replace the bacterial Pol III replicase at the replication fork, thereby disrupting DNA replication [34,44,48]. Consistent with a Pol III-Pol IV switch underlying this lethal phenotype, mutations in Pol IV that disrupt its ability to interact with either the cleft (Pol IV C ; see Table 1) or the rim (Pol IV R ) of the β clamp abrogated its ability to kill the dnaN159 strain [38]. Finally, SOS-induced levels of Pol IV modestly slowed the rate of DNA replication in vitro [13,14,45], while overproduction of Pol IV severely impeded it, possibly by replacing Pol III [13,14,45]. The finding that Pol IV LF -β clamp interactions were dispensable when Pol IV was expressed at~70-fold higher than SOS-induced levels suggested that Pol IV may interact with Pol III to effect its displacement from the β clamp [45]. Based on these results, Pol IV was suggested to act as a DNA damage checkpoint effector that acts to slow replication fork progression in response to DNA damage [13,45]. The goal of this study was to define the relationship between the physiological function of Pol IV in TLS and its ability to impede E. coli growth when overproduced. With this goal in mind, the hypersensitivity of the dnaN159 strain was exploited to identify 13 novel mutant Pol IV proteins that failed to confer lethality. Genetic and biochemical characterization of these Pol IV mutants strongly suggest that the ability of overproduced levels of Pol IV to inhibit E. coli growth is a consequence of its ability to gain inappropriate access to the replication fork via a switch that is mechanistically similar to that used under physiological conditions to coordinate the actions of Pol IV with Pol III. Importantly, further analysis of one of the mutants, Pol IV-T120P, revealed novel insights into the mechanism by which Pol IV gains access to DNA lesions in vivo. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IV R and Pol IV C , failed to support Pol IV TLS function in vivo. Using a single molecule primer extension assay, we demonstrated that the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results suggest that Pol III--Pol IV switching involves interaction of Pol IV with both Pol III and the β clamp rim and cleft regions, and provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA. | 7464696 |
Methanesulfonate | CS(=O)(=O)[O-] | E. coli Pol IV catalyzes accurate bypass of N 2 -dG lesions induced by nitrofurazone (NFZ), as well as alkylated adducts such as N 3 -methyladenine (N 3 -mdA) caused by methyl CS(=O)(=O)[O-] (MMS). As a result, E. coli strains lacking Pol IV function (i.e., ΔdinB) display sensitivity to these agents [17,[40][41][42][43]. In contrast to this protective role, overproduction of Pol IV is lethal to E. coli [34,44,45]; similarly, aberrant expression of Pol κ, the eukaryotic ortholog of dinB, promotes genome instability in human cells [46]. Lethality in aerobically cultured E. coli cells was suggested to result from toxic levels of double strand (ds) DNA breaks resulting from efforts to repair closely spaced 8-oxo-7,8-deoxyguanosine (8-oxo-dG) adducts incorporated during replication of undamaged DNA by Pol IV [47]. However, sensitivity of a dnaN159 strain to~4-fold higher than SOS-induced levels of Pol IV [34,44], and of a dnaN + E. coli strain to significantly higher than SOS-induced levels (i.e.,~70-fold; [45]), were both independent of Pol IV catalytic activity, suggesting that at least in these cases, lethality relied on one or more alternative mechanisms. The dnaN159 allele encodes a mutant form of the β sliding clamp that is deficient in regulation of proper access of the different E. coli Pols to DNA [34,35,38,44,48,49]. Thus, sensitivity of the dnaN159 strain to elevated levels of Pol IV was suggested to result from its enhanced ability to replace the bacterial Pol III replicase at the replication fork, thereby disrupting DNA replication [34,44,48]. Consistent with a Pol III-Pol IV switch underlying this lethal phenotype, mutations in Pol IV that disrupt its ability to interact with either the cleft (Pol IV C ; see Table 1) or the rim (Pol IV R ) of the β clamp abrogated its ability to kill the dnaN159 strain [38]. Finally, SOS-induced levels of Pol IV modestly slowed the rate of DNA replication in vitro [13,14,45], while overproduction of Pol IV severely impeded it, possibly by replacing Pol III [13,14,45]. The finding that Pol IV LF -β clamp interactions were dispensable when Pol IV was expressed at~70-fold higher than SOS-induced levels suggested that Pol IV may interact with Pol III to effect its displacement from the β clamp [45]. Based on these results, Pol IV was suggested to act as a DNA damage checkpoint effector that acts to slow replication fork progression in response to DNA damage [13,45]. The goal of this study was to define the relationship between the physiological function of Pol IV in TLS and its ability to impede E. coli growth when overproduced. With this goal in mind, the hypersensitivity of the dnaN159 strain was exploited to identify 13 novel mutant Pol IV proteins that failed to confer lethality. Genetic and biochemical characterization of these Pol IV mutants strongly suggest that the ability of overproduced levels of Pol IV to inhibit E. coli growth is a consequence of its ability to gain inappropriate access to the replication fork via a switch that is mechanistically similar to that used under physiological conditions to coordinate the actions of Pol IV with Pol III. Importantly, further analysis of one of the mutants, Pol IV-T120P, revealed novel insights into the mechanism by which Pol IV gains access to DNA lesions in vivo. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IV R and Pol IV C , failed to support Pol IV TLS function in vivo. Using a single molecule primer extension assay, we demonstrated that the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results suggest that Pol III--Pol IV switching involves interaction of Pol IV with both Pol III and the β clamp rim and cleft regions, and provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA. | 7464696 |
Arabinose | OC1[C@@H]([C@@H](O)[C@H](CO1)O)O | g The 937 GGG 939 !GGC substitution present in plasmid pJH154 represents a silent mutation at position G313. h The Pol IV-R75L mutant was expressed as a soluble full-length protein from the T7 promoter, but was inconsistently detected following expression in Pol IV CD from the OC1[C@@H]([C@@H](O)[C@H](CO1)O)O promoter of plasmid pDB22, suggesting Pol IV CD -R75L was misfolded. | 7464696 |
Glycine | C(O)(=O)CN | As summarized in Fig 1A, the identified Pol IV mutations are distributed throughout all 4 structural domains of Pol IV. To gain insight into the possible defect(s) associated with each mutant Pol IV, the positions of the mutations identified in each of the 13 dinB alleles were represented on in silico models for the structure of the Pol IV-β clamp complex assembled on primed DNA in either a non-replicative (meaning Pol IV is bound to both the β clamp rim and cleft, but not the DNA; see Fig 1B) or a replicative mode (meaning Pol IV is bound only to the β clamp cleft, as well as the DNA; see Fig 1C). Based upon these structural models, combined with our current understanding of E. coli Pol IV structure-function [32,53], as well as published studies of the homologous Sulfolobus solfataricus P2 Dpo4 [54,55], we inferred likely functions for several of the mutated residues in Pol IV (Table 2). Residues A15, G52, C66 and A143 likely contribute to either structural integrity or the hydrophobic core ( Fig 1A-1C), suggesting that mutations of these residues most likely alter overall Pol IV structure. Position D10 likely contributes to structural integrity and is adjacent to D8, D103 and E104 (Fig 1D), which together act to coordinate 2 Mg +2 ions to constitute the catalytic core of Pol IV, suggesting that substitution of D10 with C(O)(=O)CN might affect the structure of the Pol IV catalytic center. Residue R75 resides in the fingers domain and appears to form a hydrogen bond with residue D20 in the palm (Fig 1E), possibly contributing to the tertiary structure of Pol IV. Residues G183, G219, and R323 are all in close proximity to the DNA template and may be involved in Pol IV-DNA interactions ( Fig 1F-1H). Finally, H302 and Q342 of Pol IV are immediately adjacent to residues previously demonstrated by Bunting et al. to directly contact the β clamp rim ( 303 VWP 305 ) or cleft ( 346 QLVLGL 351 ), respectively, suggesting that the H302Q and Q342K substitutions disrupt these interactions ( [32]; Fig 1B). While presumed functions could be assigned for many of the mutants based upon previous studies, possible defects of the Pol IV mutants bearing substitutions of residues A44, T120 or A149 remain unclear ( Table 2). | 7464696 |
Arabinose | O[C@H]1C(OC[C@@H]([C@@H]1O)O)O | In order to gain insight into the relationship between the abilities of elevated levels of Pol IV to impede growth of the dnaN159 strain [34,38,44] and overproduced levels of Pol IV to kill the dnaN + strain [45], a quantitative transformation assay was used to analyze the phenotypes of pBAD derivatives bearing the relevant Pol IV mutations (see Table 1). The ability of overproduced levels of full-length Pol IV (pDB10) or the catalytic domain of Pol IV (Pol IV CD ; residues 1-230 expressed from pDB12) to impede growth of E. coli was independent of both its catalytic activity [45] and aerobic growth (S4 Fig), similar to the situation discussed above for the dnaN159 strain (S1 Table and S2 Table). Since overexpression of Pol IV CD was necessary and sufficient to impede growth ( [45]; see Fig 2), we focused on Pol IV mutations mapping within the first 230 residues of Pol IV. Despite the fact that the mutant Pol IV proteins appeared stable when expressed from their native dinB promoter contained within a low copy number plasmid (S1 Fig), 6 of the 11 mutants containing substitutions within the first 230 residues of Pol IV displayed either poor solubility or signs of extensive proteolysis following their overproduction from the T7 promoter while one mutant (R75L) was specifically unstable when cloned into Pol IV CD -expressing pBAD plasmid (see Table 2). These Pol IV mutants were not pursued further. Results for the other 4 Pol IV mutants are summarized in Fig 2. The plasmids overproducing Pol IV CD -D10G (pDB20), Pol IV CD -C66S (pDB21), Pol IV CD -T120P (pDB23), or Pol IV CD -G183V (pDB25) each transformed the dnaN + strain with an efficiency comparable to that of dinB Mutants Impaired for Lesion Bypass In Vivo the pBAD control, both in the presence or absence of O[C@H]1C(OC[C@@H]([C@@H]1O)O)O (albeit most exhibited tiny to small colonies), while the plasmid overproducing wild type Pol IV (pDB10) or Pol IV CD (pDB12) failed to transform in the presence of O[C@H]1C(OC[C@@H]([C@@H]1O)O)O (Fig 2). However, in contrast to the other mutants, which formed tiny to small colonies in the presence of O[C@H]1C(OC[C@@H]([C@@H]1O)O)O (Fig 2), the strain overproducing Pol IV CD -T120P displayed robust colonies that were indistinguishable from the strain bearing either the empty pBAD plasmid or overproducing Pol IV LF (pDB14). In contrast to the robust growth observed for the strain overproducing Pol IV CD -T120P, the strain overproducing the full length Pol IV-T120P (pDB33) failed to grow in the presence of O[C@H]1C(OC[C@@H]([C@@H]1O)O)O (Fig 2). We confirmed that the Pol IV CD -T120P mutant was expressed in a soluble form and at a level comparable to wild type Pol IV CD (see legend to Fig 2). Based on this observation and results discussed later in this report, the difference between the growth phenotype of the strain expressing Pol IV CD -T120P and that expressing full length Pol IV-T120P appears to be a result of the Pol IV LF domain, which contributes to the ability of Pol IV to impede E. coli growth [16,45]. Taken together, these findings suggest that a common mechanism underlies the ability of overproduced levels of Pol IV CD to impede growth of the dnaN + strain and near-physiological levels of Pol IV to impede growth of the dnaN159 strain. Furthermore, the fact that Pol IV CD lacks the β clamp-binding Pol IV LF domain, yet is nevertheless able to displace Pol III from the β clamp in vitro [16,45], suggests that Pol IV CD interacts physically with one or more subunit of Pol III holoenzyme. Thus, the ability of T120P to alleviate the lethal phenotype may be indicative of this mutant being impaired for a Pol III-Pol IV interaction. | 7464696 |
Glycine | C(=O)(O)CN | In the absence of accessory proteins, Pol IV R , Pol IV C , Pol IV-T120P and Pol IV-H302Q/ Q342K exhibited replication activity roughly comparable to wild type Pol IV (Fig 3A). In contrast, Pol IV-R323S was only modestly active, while Pol IV-D10G, Pol IV-C66S, Pol IV-R75L and Pol IV-G183V lacked detectable activity, similar to Pol IV-D103N (Fig 3A). In the presence of SSB, β clamp and the DnaX complex, Pol IV R and Pol IV-T120P were again indistinguishable from wild type Pol IV (Fig 3B). Pol IV-C66S, Pol IV-R75L, Pol IV-G183V and Pol IV-R323S each displayed modest replication activity (Fig 3B), suggesting that the presence of accessory factors compensated in part for their intrinsic biochemical defects, possibly by helping to recruit the mutant Pol IV proteins to the primer/template junction, and/or by stabilizing an active conformation of the mutant Pol IV protein. Whereas Pol IV-H302Q/Q342K was indistinguishable from wild type Pol IV in the absence of accessory proteins (Fig 3A), it was impaired for processive replication in their presence compared to wild type (Fig 3B), suggesting that the Q342K mutation, which is adjacent to the Pol IV CBM (see Fig 1), interferes with the Pol IV-β clamp cleft interaction. Finally, Pol IV-D10G lacked detectable activity, similar to the D103N mutation, suggesting that D10 either participates directly in catalysis, or its substitution with C(=O)(O)CN perturbs the structure of the catalytic center (see Fig 1D). Taken together, these results demonstrate that with the exception of Pol IV-D10G, each of the mutant proteins retained at least partial catalytic activity in vitro. Remarkably, Pol IV-T120P supported replication activity and processivity that were each comparable to that of wild type Pol IV. | 7464696 |
Proline | N1CCC[C@H]1C(=O)O | In order to quantify the replication activity of Pol IV-T120P more rigorously, we measured its kinetic parameters and compared them to those of wild type Pol IV. As summarized in Table 3, the catalytic efficiency (k pol /K d ) of Pol IV-T120P was~2.5-fold higher than wild type Pol IV for incorporation of dC opposite template dG, and~0.5-fold lower than wild type Pol IV for incorporation of dT opposite template dA. Both Pol IV and Pol IV-T120P were able to incorporate the other three dNTPs opposite a template dG or dA. However, in all cases the efficiency of misincorporation was significantly less (<10%) than that measured for correct incorporation. The small differences in catalytic efficiency between wild type Pol IV and Pol IV-T120P were attributable to effects of the T120P substitution on both dNTP binding (K d ) and Pol turnover (k pol ) (Table 3). Thus, despite the fact that residue T120 is well removed from the catalytic center of Pol IV (Fig 1), its substitution with a N1CCC[C@H]1C(=O)O nevertheless exerts a modest effect on Pol IV catalysis. These findings, taken together with those discussed above, confirm that Pol IV-T120P retains essentially wild type Pol activity when replicating undamaged DNA, despite its inability to impede E. coli growth when expressed at elevated levels. | 7464696 |
Mimic | C1=C(C)C=C(C)C=C1C(N(NC(C1C=CC(CC)=CC=1)=O)C(C)(C)C)=O | To determine if the MMS phenotypes of the Pol IV-T120P strain were due to a catalytic TLS defect which rendered Pol IV-T120P incapable of bypassing MMS-induced lesions, we used a primer extension assay to measure the ability of purified Pol IV-T120P to catalyze in vitro bypass of the model MMS-induced lesions O 6 -methylguanine (O 6 -mdG), 3-deaza-3-methyladenine (3d-medA), which is a stable C1=C(C)C=C(C)C=C1C(N(NC(C1C=CC(CC)=CC=1)=O)C(C)(C)C)=O of N 3 -mdA [59], as well as an AP site. As summarized in Table 3, both wild type Pol IV and Pol IV-T120P were able to bypass template O 6 -mdG and 3d-medA. While there were some differences in substrate binding (K d ) and/or turnover (k pol ), Pol IV-T120P was as efficient or more so than wild type Pol IV. Pol IV and Pol IV-T120P each incorporated either dC or dT opposite template O 6 -mdG with roughly equivalent efficiencies. In both cases, bypass was considerably less efficient than that observed for template dG, due to a reduction in both K d and k pol , with Pol IV-T120P slightly outperforming wild type Pol IV. Both Pols were capable of incorporating low levels of dA or dG opposite template O 6 -mdG. However, the amount of incorporation was less than 10% compared to that for the insertion of dC or dT opposite the alkylated lesion. 3d-medA was easier for both Pol IV and Pol IV-T120P to bypass, again with Pol IV-T120 outperforming wild type Pol IV by a factor of~2-fold, attributable in large part to stronger substrate binding (K d ). Both Pols incorporated dA, dC or dG opposite template 3d-medA. The level of incorporation was significantly less than that measured for incorporation of dT. Finally, even though Pol IV-T120P was marginal in regard to its ability to bypass the AP site, inserting dA, it was nevertheless more efficient than wild type Pol IV (Table 3). Taken together, these results demonstrate that Pol IV-T120P is proficient in vitro for TLS past a variety of DNA adducts commonly induced by MMS, suggesting that the inability of Pol IV-T120P to cope with MMS-induced DNA damage in vivo was the result of its inability to gain access to the lesions. | 7464696 |
Arabinose | [C@@H]1(O)[C@@H](O)C(O)OC[C@@H]1O | We previously utilized a single molecule primer extension assay to demonstrate exchange of Pol III and Pol IV on β clamp at the 3' primer terminus in vitro [12]. The distinct polymerization rates of Pol III and Pol IV allowed us to unambiguously assign individual DNA synthesis events to each respective Pol and to measure their respective processivities when incubated alone or together (Fig 7A). At 300 nM Pol IV, a 60-fold molar excess over Pol III that simulates levels found in SOS-induced cells [3], Pol IV actively displaced Pol III from the DNA template as inferred from Pol III processivity measurements ( [12,16,17,38,45]; see Fig 7B). This ability of Pol IV to reduce Pol III processivity was dependent on the Pol IV CBM, arguing that Pol III displacement involves at a minimum a conformational exchange of the two Pols on the β clamp. Using this approach, we asked whether Pol IV-T120P was likewise able to displace Pol III from the β clamp, as inferred by a reduction in its processivity. As summarized in Fig 7B, a 60-fold molar excess of Pol IV-T120P over Pol III was as efficient as wild type Pol IV at inhibiting Pol III processivity. Together, these results suggest one of two possibilities: either (i) the T120P mutation does not impact the ability of Pol IV to displace Pol III from β clamp, or (ii) efficient recruitment of Pol IV to the Pol III-β clamp complex through its interactions with β clamp masks the Pol IV-T120P-dependent defect in Pol IV displacement of Pol III from β clamp. To distinguish between these two models, we analyzed the ability of Pol IV CD and Pol IV CD -T120P, both of which lack the Pol IV LF β clamp-binding domain, to impede Pol III processivity using the same single molecule assay. Furukohri and colleagues previously demonstrated that a~900-to 1,800-fold molar excess of Pol IV CD over Pol III (890 nM Pol IV compared to 0.5-1.0 nM Pol III) was able to disrupt the Pol III-β clamp complex assembled in dinB Mutants Impaired for Lesion Bypass In Vivo vitro on a primed DNA substrate [16]. Similarly, we found that 900 nM Pol IV CD (a 180-fold molar excess over Pol III) disrupted Pol III synthesis, reducing its processivity to one-half of that observed in the absence of Pol IV CD (Fig 7C, p <0.01, determined using the Wilcoxon rank-sum test). This reduction in Pol III processivity most likely results from the ability of Pol IV CD to displace Pol III from the β clamp assembled on DNA [12,16,45]. Importantly, an equivalent concentration of Pol IV CD -T120P failed to reduce processivity of Pol III. Taken together, these findings support the view that residue T120 of Pol IV plays an important role in displacing Pol III from the β clamp, and demonstrate that the Pol IV LF domain contributes to this ability. Finally, these biochemical results are remarkably similar to the growth phenotypes observed for strains overproducing Pol IV CD -T120P (pDB23) or full length Pol IV-T120P (pDB33) from the [C@@H]1(O)[C@@H](O)C(O)OC[C@@H]1O promoter (Fig 2), which demonstrate the ability of the Pol IV LF domain to mask the phenotype of the T120P mutation in vivo. | 7464696 |
Arabinose | [C@@H]1(C(O)OC[C@H](O)[C@@H]1O)O | With the goal of gaining new insights into the relationship between the physiological function of Pol IV in TLS and its ability when overexpressed to impede E. coli growth, we exploited the hypersensitivity of the dnaN159 strain to elevated levels of Pol IV to identify 13 novel Pol IV mutants that were unable to impede growth ( Table 2). These Pol IV mutants were deficient in stimulating reversion of the CC108 lacZ -1 frameshift reporter when expressed at SOS-induced levels (S2 Fig), and for conferring UV sensitivity upon the dnaN159 strain (S3 Fig), indicating that they were unable to effectively compete with Pol III for access to the replication fork. Likewise, these mutations failed to impede growth of the dnaN + strain when introduced into Pol IV CD and overproduced from the [C@@H]1(C(O)OC[C@H](O)[C@@H]1O)O promoter (Fig 2). Finally, despite the fact that all but one of the mutant Pol IV proteins (Pol IV-D10G) retained appreciable catalytic activity in vitro (Fig 3), they were nevertheless impaired for tolerating MMS-induced lesions in vivo (Fig 4). These results, taken together with those discussed below, support the view that overexpressed levels of Pol IV impede E. coli growth by actively replacing Pol III at the replication fork via a mechanism that is similar to that used under physiological conditions to coordinate high fidelity processive Pol III replication with potentially mutagenic Pol IV TLS. In contrast to an earlier study [47], we failed to observe an ability of overproduced levels of Pol IV to mediate cell death in either the dnaN159 or dnaN + strains via excessive incorporation of oxidized precursors (S2 Table and S4 Fig). Thus, Pol IV appears to be able to impede E. coli growth by either incorporating lethal levels of 8-oxo-dG or by displacing Pol III. This view is consistent with the finding that under the conditions used in this study lethality was independent of Pol IV catalytic activity [38,45]. However, our finding that several Pol IV mutants identified in this work were impaired for catalytic activity in vitro (Fig 3) suggests that the ability of Pol IV to replace Pol III at the replication fork is dependent at least in part on residues in Pol IV that contribute to catalytic activity. Alternatively, the ability of Pol IV to persist at the replication fork after replacing Pol III likely contributes to the killing, and would rely on Pol IV processivity, which, with the exception of Pol IV-T120P, was impaired in the Pol IV mutants analyzed here. | 7464696 |
Proline | C1N[C@@H](CC1)C(=O)O | Results discussed in this report provide several new insights into the mechanism by which the actions of Pol III are coordinately regulated with those of Pol IV, and when taken together with previously published findings [17,33,38,67], support a new model for the role of Pol III--Pol IV switching in Pol IV-mediated TLS in vivo. In addition to its interactions with the β clamp, an interaction of Pol IV with Pol III also appears to play a biologically important role in recruiting Pol IV to lesions (Figs 5 and 6). Biochemical interaction of Pol IV CD with Pol III holoenzyme is sufficient to mediate displacement of Pol III from the face of the β clamp in vitro [12,13,16,45]. Our single molecule assay reproduced this finding, and further demonstrated that position T120 of Pol IV is important for this function (Fig 7). Residue T120 is one helical turn from the start of α-helix 5 (see Fig 8) and its substitution with C1N[C@@H](CC1)C(=O)O likely [84]. Pol IV (PDB 4IR9) was docked onto the rim of the β clamp in complex with Pol IIIαεθ by aligning it with the Pol IV LF domain in PDB 1UNN [12,53]. Residue T120 of Pol IV is in orange, while α-helix 5 corresponding to residues H116-Q135 of Pol IV is in yellow. (C) Model of the Pol III-Pol IV switching mechanism in which Pol IV (cyan) gains access to the DNA template after making contact with one or more subunits of Pol III (green), as well as the rim of the β clamp (purple). The small black circles represent the clefts in the β clamp. See text for further details. | 7464696 |
Thiamine | CC1=C(SC=[N+]1CC1C(N)=NC(=NC=1)C)CCO | Bacteria were cultured in either Luria Bertani (LB; 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), or M9 minimal medium (12.9 g/L Na 2 HPO 4 •7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl) supplemented with 0.1 mM CaCl 2 , 2 mM MgCl 2 , 5 μg/ml CC1=C(SC=[N+]1CC1C(N)=NC(=NC=1)C)CCO, 0.5% casamino acids and 0.5% glucose or 0.2% arabinose, as indicated. For anaerobic growth, 100 mM KNO 3 was added to the growth to act as the terminal electron acceptor. When required, the following antibiotics were used at the indicated concentrations: ampicillin (Amp), 150 μg/ml; tetracycline (Tet), 10 μg/ml; kanamycin (Kan), 40 μg/ml; chloramphenicol (Cam), 20 μg/ml; rifampicin (Rif), 50 μg/ml. E. coli strains were constructed using P1vir-mediated generalized transduction [73], or λRed-mediated recombineering [74], and are described in Table 1. Strain genotypes were verified using either diagnostic PCR or nucleotide sequence analysis (Roswell Park Biopolymer Facility, Buffalo, NY) of respective PCR-amplified alleles. Strains were made competent for transformation using CaCl 2 as described [48]. Bacterial plasmid transformation frequency [38], UV sensitivity [38,48] and lacZ!Lac + reversion [51,52,56] was measured as described in the indicated references. Plasmid DNAs are described in Table 1. Standard techniques were used for cloning. Site-directed mutagenesis was performed using the Quickchange kit (Stratagene). Synthetic oligonucleotide primers used for mutagenesis were purchased from either IDT or Operon, and their sequences are presented in S3 Table. All plasmid sequences were confirmed by nucleotide sequence (Roswell Park Biopolymer Facility, Buffalo, NY). Mutant dinB alleles were subcloned from pWSK29 into pET11a (Novagen) by NdeI and BamHI (Fermentas) restriction, followed by ligation to the similarly prepared pET11a backbone using T4 DNA ligase (Fermentas). Sensitivity to MMS (Sigma) was measured as described [41]. | 7464696 |
Arabinose | [C@@H]1(COC([C@H](O)[C@H]1O)O)O | Bacteria were cultured in either Luria Bertani (LB; 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), or M9 minimal medium (12.9 g/L Na 2 HPO 4 •7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl) supplemented with 0.1 mM CaCl 2 , 2 mM MgCl 2 , 5 μg/ml thiamine, 0.5% casamino acids and 0.5% glucose or 0.2% [C@@H]1(COC([C@H](O)[C@H]1O)O)O, as indicated. For anaerobic growth, 100 mM KNO 3 was added to the growth to act as the terminal electron acceptor. When required, the following antibiotics were used at the indicated concentrations: ampicillin (Amp), 150 μg/ml; tetracycline (Tet), 10 μg/ml; kanamycin (Kan), 40 μg/ml; chloramphenicol (Cam), 20 μg/ml; rifampicin (Rif), 50 μg/ml. E. coli strains were constructed using P1vir-mediated generalized transduction [73], or λRed-mediated recombineering [74], and are described in Table 1. Strain genotypes were verified using either diagnostic PCR or nucleotide sequence analysis (Roswell Park Biopolymer Facility, Buffalo, NY) of respective PCR-amplified alleles. Strains were made competent for transformation using CaCl 2 as described [48]. Bacterial plasmid transformation frequency [38], UV sensitivity [38,48] and lacZ!Lac + reversion [51,52,56] was measured as described in the indicated references. Plasmid DNAs are described in Table 1. Standard techniques were used for cloning. Site-directed mutagenesis was performed using the Quickchange kit (Stratagene). Synthetic oligonucleotide primers used for mutagenesis were purchased from either IDT or Operon, and their sequences are presented in S3 Table. All plasmid sequences were confirmed by nucleotide sequence (Roswell Park Biopolymer Facility, Buffalo, NY). Mutant dinB alleles were subcloned from pWSK29 into pET11a (Novagen) by NdeI and BamHI (Fermentas) restriction, followed by ligation to the similarly prepared pET11a backbone using T4 DNA ligase (Fermentas). Sensitivity to MMS (Sigma) was measured as described [41]. | 7464696 |
Ampicillin | [C@@H]1([C@H]2SC(C)(C)[C@@H](N2C1=O)C(O)=O)NC(=O)[C@@H](c1ccccc1)N | Bacteria were cultured in either Luria Bertani (LB; 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), or M9 minimal medium (12.9 g/L Na 2 HPO 4 •7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl) supplemented with 0.1 mM CaCl 2 , 2 mM MgCl 2 , 5 μg/ml thiamine, 0.5% casamino acids and 0.5% glucose or 0.2% arabinose, as indicated. For anaerobic growth, 100 mM KNO 3 was added to the growth to act as the terminal electron acceptor. When required, the following antibiotics were used at the indicated concentrations: [C@@H]1([C@H]2SC(C)(C)[C@@H](N2C1=O)C(O)=O)NC(=O)[C@@H](c1ccccc1)N (Amp), 150 μg/ml; tetracycline (Tet), 10 μg/ml; kanamycin (Kan), 40 μg/ml; chloramphenicol (Cam), 20 μg/ml; rifampicin (Rif), 50 μg/ml. E. coli strains were constructed using P1vir-mediated generalized transduction [73], or λRed-mediated recombineering [74], and are described in Table 1. Strain genotypes were verified using either diagnostic PCR or nucleotide sequence analysis (Roswell Park Biopolymer Facility, Buffalo, NY) of respective PCR-amplified alleles. Strains were made competent for transformation using CaCl 2 as described [48]. Bacterial plasmid transformation frequency [38], UV sensitivity [38,48] and lacZ!Lac + reversion [51,52,56] was measured as described in the indicated references. Plasmid DNAs are described in Table 1. Standard techniques were used for cloning. Site-directed mutagenesis was performed using the Quickchange kit (Stratagene). Synthetic oligonucleotide primers used for mutagenesis were purchased from either IDT or Operon, and their sequences are presented in S3 Table. All plasmid sequences were confirmed by nucleotide sequence (Roswell Park Biopolymer Facility, Buffalo, NY). Mutant dinB alleles were subcloned from pWSK29 into pET11a (Novagen) by NdeI and BamHI (Fermentas) restriction, followed by ligation to the similarly prepared pET11a backbone using T4 DNA ligase (Fermentas). Sensitivity to MMS (Sigma) was measured as described [41]. | 7464696 |
Tetracycline | C1(=O)C(=C([C@@]2([C@H]([C@@H]1N(C)C)C[C@@H]1[C@](C3=C(C(=C1C2=O)O)C(=CC=C3)O)(C)O)O)O)C(N)=O | Bacteria were cultured in either Luria Bertani (LB; 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), or M9 minimal medium (12.9 g/L Na 2 HPO 4 •7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl) supplemented with 0.1 mM CaCl 2 , 2 mM MgCl 2 , 5 μg/ml thiamine, 0.5% casamino acids and 0.5% glucose or 0.2% arabinose, as indicated. For anaerobic growth, 100 mM KNO 3 was added to the growth to act as the terminal electron acceptor. When required, the following antibiotics were used at the indicated concentrations: ampicillin (Amp), 150 μg/ml; C1(=O)C(=C([C@@]2([C@H]([C@@H]1N(C)C)C[C@@H]1[C@](C3=C(C(=C1C2=O)O)C(=CC=C3)O)(C)O)O)O)C(N)=O (Tet), 10 μg/ml; kanamycin (Kan), 40 μg/ml; chloramphenicol (Cam), 20 μg/ml; rifampicin (Rif), 50 μg/ml. E. coli strains were constructed using P1vir-mediated generalized transduction [73], or λRed-mediated recombineering [74], and are described in Table 1. Strain genotypes were verified using either diagnostic PCR or nucleotide sequence analysis (Roswell Park Biopolymer Facility, Buffalo, NY) of respective PCR-amplified alleles. Strains were made competent for transformation using CaCl 2 as described [48]. Bacterial plasmid transformation frequency [38], UV sensitivity [38,48] and lacZ!Lac + reversion [51,52,56] was measured as described in the indicated references. Plasmid DNAs are described in Table 1. Standard techniques were used for cloning. Site-directed mutagenesis was performed using the Quickchange kit (Stratagene). Synthetic oligonucleotide primers used for mutagenesis were purchased from either IDT or Operon, and their sequences are presented in S3 Table. All plasmid sequences were confirmed by nucleotide sequence (Roswell Park Biopolymer Facility, Buffalo, NY). Mutant dinB alleles were subcloned from pWSK29 into pET11a (Novagen) by NdeI and BamHI (Fermentas) restriction, followed by ligation to the similarly prepared pET11a backbone using T4 DNA ligase (Fermentas). Sensitivity to MMS (Sigma) was measured as described [41]. | 7464696 |
Kanamycin | [C@@H]1(C[C@@H](N)[C@H](O[C@H]2O[C@H](CO)[C@H]([C@@H]([C@H]2O)N)O)[C@@H](O)[C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CN)O1)O)N | Bacteria were cultured in either Luria Bertani (LB; 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), or M9 minimal medium (12.9 g/L Na 2 HPO 4 •7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl) supplemented with 0.1 mM CaCl 2 , 2 mM MgCl 2 , 5 μg/ml thiamine, 0.5% casamino acids and 0.5% glucose or 0.2% arabinose, as indicated. For anaerobic growth, 100 mM KNO 3 was added to the growth to act as the terminal electron acceptor. When required, the following antibiotics were used at the indicated concentrations: ampicillin (Amp), 150 μg/ml; tetracycline (Tet), 10 μg/ml; [C@@H]1(C[C@@H](N)[C@H](O[C@H]2O[C@H](CO)[C@H]([C@@H]([C@H]2O)N)O)[C@@H](O)[C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CN)O1)O)N (Kan), 40 μg/ml; chloramphenicol (Cam), 20 μg/ml; rifampicin (Rif), 50 μg/ml. E. coli strains were constructed using P1vir-mediated generalized transduction [73], or λRed-mediated recombineering [74], and are described in Table 1. Strain genotypes were verified using either diagnostic PCR or nucleotide sequence analysis (Roswell Park Biopolymer Facility, Buffalo, NY) of respective PCR-amplified alleles. Strains were made competent for transformation using CaCl 2 as described [48]. Bacterial plasmid transformation frequency [38], UV sensitivity [38,48] and lacZ!Lac + reversion [51,52,56] was measured as described in the indicated references. Plasmid DNAs are described in Table 1. Standard techniques were used for cloning. Site-directed mutagenesis was performed using the Quickchange kit (Stratagene). Synthetic oligonucleotide primers used for mutagenesis were purchased from either IDT or Operon, and their sequences are presented in S3 Table. All plasmid sequences were confirmed by nucleotide sequence (Roswell Park Biopolymer Facility, Buffalo, NY). Mutant dinB alleles were subcloned from pWSK29 into pET11a (Novagen) by NdeI and BamHI (Fermentas) restriction, followed by ligation to the similarly prepared pET11a backbone using T4 DNA ligase (Fermentas). Sensitivity to MMS (Sigma) was measured as described [41]. | 7464696 |
Chloramphenicol | [C@H](CO)(NC(=O)C(Cl)Cl)[C@H](O)c1ccc([N+](=O)[O-])cc1 | Bacteria were cultured in either Luria Bertani (LB; 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), or M9 minimal medium (12.9 g/L Na 2 HPO 4 •7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl) supplemented with 0.1 mM CaCl 2 , 2 mM MgCl 2 , 5 μg/ml thiamine, 0.5% casamino acids and 0.5% glucose or 0.2% arabinose, as indicated. For anaerobic growth, 100 mM KNO 3 was added to the growth to act as the terminal electron acceptor. When required, the following antibiotics were used at the indicated concentrations: ampicillin (Amp), 150 μg/ml; tetracycline (Tet), 10 μg/ml; kanamycin (Kan), 40 μg/ml; [C@H](CO)(NC(=O)C(Cl)Cl)[C@H](O)c1ccc([N+](=O)[O-])cc1 (Cam), 20 μg/ml; rifampicin (Rif), 50 μg/ml. E. coli strains were constructed using P1vir-mediated generalized transduction [73], or λRed-mediated recombineering [74], and are described in Table 1. Strain genotypes were verified using either diagnostic PCR or nucleotide sequence analysis (Roswell Park Biopolymer Facility, Buffalo, NY) of respective PCR-amplified alleles. Strains were made competent for transformation using CaCl 2 as described [48]. Bacterial plasmid transformation frequency [38], UV sensitivity [38,48] and lacZ!Lac + reversion [51,52,56] was measured as described in the indicated references. Plasmid DNAs are described in Table 1. Standard techniques were used for cloning. Site-directed mutagenesis was performed using the Quickchange kit (Stratagene). Synthetic oligonucleotide primers used for mutagenesis were purchased from either IDT or Operon, and their sequences are presented in S3 Table. All plasmid sequences were confirmed by nucleotide sequence (Roswell Park Biopolymer Facility, Buffalo, NY). Mutant dinB alleles were subcloned from pWSK29 into pET11a (Novagen) by NdeI and BamHI (Fermentas) restriction, followed by ligation to the similarly prepared pET11a backbone using T4 DNA ligase (Fermentas). Sensitivity to MMS (Sigma) was measured as described [41]. | 7464696 |
Rifampicin | [C@H]1([C@H](O)[C@@H]([C@H]([C@@H](C)/C=C\C=C(\C)C(Nc2c(C=NN3CCN(C)CC3)c(O)c3c(c2O)c(O)c(c2c3C([C@](C)(O2)O/C=C/[C@H](OC)[C@H]([C@H]1OC(C)=O)C)=O)C)=O)O)C)C | Bacteria were cultured in either Luria Bertani (LB; 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), or M9 minimal medium (12.9 g/L Na 2 HPO 4 •7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl) supplemented with 0.1 mM CaCl 2 , 2 mM MgCl 2 , 5 μg/ml thiamine, 0.5% casamino acids and 0.5% glucose or 0.2% arabinose, as indicated. For anaerobic growth, 100 mM KNO 3 was added to the growth to act as the terminal electron acceptor. When required, the following antibiotics were used at the indicated concentrations: ampicillin (Amp), 150 μg/ml; tetracycline (Tet), 10 μg/ml; kanamycin (Kan), 40 μg/ml; chloramphenicol (Cam), 20 μg/ml; [C@H]1([C@H](O)[C@@H]([C@H]([C@@H](C)/C=C\C=C(\C)C(Nc2c(C=NN3CCN(C)CC3)c(O)c3c(c2O)c(O)c(c2c3C([C@](C)(O2)O/C=C/[C@H](OC)[C@H]([C@H]1OC(C)=O)C)=O)C)=O)O)C)C (Rif), 50 μg/ml. E. coli strains were constructed using P1vir-mediated generalized transduction [73], or λRed-mediated recombineering [74], and are described in Table 1. Strain genotypes were verified using either diagnostic PCR or nucleotide sequence analysis (Roswell Park Biopolymer Facility, Buffalo, NY) of respective PCR-amplified alleles. Strains were made competent for transformation using CaCl 2 as described [48]. Bacterial plasmid transformation frequency [38], UV sensitivity [38,48] and lacZ!Lac + reversion [51,52,56] was measured as described in the indicated references. Plasmid DNAs are described in Table 1. Standard techniques were used for cloning. Site-directed mutagenesis was performed using the Quickchange kit (Stratagene). Synthetic oligonucleotide primers used for mutagenesis were purchased from either IDT or Operon, and their sequences are presented in S3 Table. All plasmid sequences were confirmed by nucleotide sequence (Roswell Park Biopolymer Facility, Buffalo, NY). Mutant dinB alleles were subcloned from pWSK29 into pET11a (Novagen) by NdeI and BamHI (Fermentas) restriction, followed by ligation to the similarly prepared pET11a backbone using T4 DNA ligase (Fermentas). Sensitivity to MMS (Sigma) was measured as described [41]. | 7464696 |
Agarose | C1[C@@H]2O[C@H]([C@@H](O)[C@H](O1)[C@@H]2O[C@@H]1O[C@H](CO)[C@@H]([C@@H]([C@H]1O)O)O)O[C@H]1[C@H]([C@H](O[C@H]2[C@@H]3[C@@H]([C@@H](O[C@H]2CO3)O)O)O[C@@H]([C@@H]1O)CO)O | Spontaneous dinB mutations unable to impede growth of the dnaN159 lexA51(Def) E. coli strain MS105 were identified by selecting Amp R transformants using 200 ng of plasmid pJH110. This strain displayed a transformation efficiency of~10 6 colony forming units/μg of supercoiled pWSK29 plasmid DNA. In instances where multiple colonies were obtained from a single transformation reaction, a single CFU was selected from the plate for further analysis to avoid sibling mutations. Plasmids were isolated using the Qiagen mini-prep kit as per the manufacturer's recommendation. Purified plasmids were analyzed by C1[C@@H]2O[C@H]([C@@H](O)[C@H](O1)[C@@H]2O[C@@H]1O[C@H](CO)[C@@H]([C@@H]([C@H]1O)O)O)O[C@H]1[C@H]([C@H](O[C@H]2[C@@H]3[C@@H]([C@@H](O[C@H]2CO3)O)O)O[C@@H]([C@@H]1O)CO)O gel electrophoresis. Those of the appropriate size were retransformed into MS105 to verify their inability to impede growth. Those that transformed MS105 with an efficiency similar to that of the pWSK29 control plasmid were then analyzed by Western blotting using polyclonal anti-Pol IV antibodies as described [4,17]. | 7464696 |
Chloramphenicol | [O-][N+](=O)c1ccc(cc1)[C@H]([C@@H](CO)NC(C(Cl)Cl)=O)O | Strains MKS103-MKS107 were constructed using λ recombineering as described [74]. Briefly, the 2,329 bp lafU' zaf-3633::cat dinB80 yafN' DNA cassette was PCR amplified from plasmid pMKS100, pMKS101, pMKS102, pMKS103 or pMKS104 using primers P1 and P4 (S3 Table), and electroporated into E. coli strain MG1655 containing pKD46 as described [75]. [O-][N+](=O)c1ccc(cc1)[C@H]([C@@H](CO)NC(C(Cl)Cl)=O)O resistant colonies were selected on LB agar plates supplemented with [O-][N+](=O)c1ccc(cc1)[C@H]([C@@H](CO)NC(C(Cl)Cl)=O)O, and subsequently confirmed to contain the desired dinB allele by diagnostic PCR using primers MKS055 and MKS046, which anneal 589 bp upstream of primer P1 and 498 bp downstream of primer P4, respectively. The remaining primers listed in S3 Table were used for nucleotide sequence verification of the lafU'-zaf-3633::cat-dinB-yafN' cassette from 91 bp upstream of the start of primer P1 to 163 bp downstream from the end of primer P4, except for a 153 bp internal segment of the cat gene corresponding to amino acid residues L45-D96, prior to using P1vir to transduce the linked zaf-3633::cat and dinB alleles into a fresh isolate of strain MG1655. | 7464696 |
Tris-HCl | OCC(N)(CO)CO.Cl | Wild type and Pol IV mutant proteins [34], SSB [76], the γ 3 δδ' form of the DnaX clamp loader [36] and β clamp [77] were purified as described in the indicated references. Primer extension assays were performed as described previously [17,34,56] using the 32 P-radiolabeled PAGE purified 30-mer/100-mer DNA template. Briefly, reactions (20 μl) contained replication buffer (20 mM OCC(N)(CO)CO.Cl [pH 7.5], 8.0 mM MgCl 2 , 0.1 mM EDTA, 5 mM DTT, 1 mM ATP, 5% glycerol, and 0.8 μg/ml BSA), 1 nM 30-mer/100-mer template, 133 μM dNTPs (Fermentas), 90 nM SSB, 1 nM γ 3 δδ' DnaX clamp loader complex, 10 nM β clamp and 1 nM Pol IV. The reactions were pre-incubated for 3 min at 37°C to permit loading of β clamp prior to initiating replication by addition of dNTPs. Reactions were next incubated at 37°C for 5 min, then quenched by the addition of 25 mM EDTA and incubation at 95°C for 3 minutes. Aliquots of each reaction were then electrophoresed through an 8% UREA-PAGE at 60 watts for 3,332 volt hours, as described [56]. Replication products were visualized using a Bio-Rad imaging screen K and a Bio-Rad Personal Molecular Imager FX. | 7464696 |
Glycerol | C(O)(CO)CO | Wild type and Pol IV mutant proteins [34], SSB [76], the γ 3 δδ' form of the DnaX clamp loader [36] and β clamp [77] were purified as described in the indicated references. Primer extension assays were performed as described previously [17,34,56] using the 32 P-radiolabeled PAGE purified 30-mer/100-mer DNA template. Briefly, reactions (20 μl) contained replication buffer (20 mM Tris-HCl [pH 7.5], 8.0 mM MgCl 2 , 0.1 mM EDTA, 5 mM DTT, 1 mM ATP, 5% C(O)(CO)CO, and 0.8 μg/ml BSA), 1 nM 30-mer/100-mer template, 133 μM dNTPs (Fermentas), 90 nM SSB, 1 nM γ 3 δδ' DnaX clamp loader complex, 10 nM β clamp and 1 nM Pol IV. The reactions were pre-incubated for 3 min at 37°C to permit loading of β clamp prior to initiating replication by addition of dNTPs. Reactions were next incubated at 37°C for 5 min, then quenched by the addition of 25 mM EDTA and incubation at 95°C for 3 minutes. Aliquots of each reaction were then electrophoresed through an 8% UREA-PAGE at 60 watts for 3,332 volt hours, as described [56]. Replication products were visualized using a Bio-Rad imaging screen K and a Bio-Rad Personal Molecular Imager FX. | 7464696 |
Digoxigenin | C1[C@H]2[C@@](CC[C@@H](C2)O)(C)[C@H]2C[C@H]([C@]3([C@](CC[C@@H]3C3=CC(=O)OC3)([C@@H]2C1)O)C)O | Primer extension by Pol III and Pol IV was observed on single DNA molecules within custom microfluidic flow cells, as previously described [12]. Briefly, primed, single-stranded DNA substrates were derived from 7.2 kb phage M13mp18 DNA (New England Biolabs) end-labeled with C1[C@H]2[C@@](CC[C@@H](C2)O)(C)[C@H]2C[C@H]([C@]3([C@](CC[C@@H]3C3=CC(=O)OC3)([C@@H]2C1)O)C)O and biotin. DNAs were bound to the streptavidin-coated flow cell surface on one end, and to anti-C1[C@H]2[C@@](CC[C@@H](C2)O)(C)[C@H]2C[C@H]([C@]3([C@](CC[C@@H]3C3=CC(=O)OC3)([C@@H]2C1)O)C)O-coupled 2.8 μm-diameter beads on the other. Laminar flow through the flow cell exerted a constant~3 pN force on the bead, and, by extension, uniformly throughout the tether. Conversion of entropically coiled ssDNA to extended dsDNA by a Pol at this constant force was observed as motion of the bead using dark-field microscopy. This cutoff captured~95% of events in experiments with each polymerase alone. The Pol III replisome components used in the single molecule experiments were purified as previously described: β [80]; α, δ and δ' [81]; ε and θ [82]; and τ and χψ [83]. The Pol III core αεθ and the clamp loader assembly with stoichiometry τ 3 δδ'χψ were then assembled and purified [83]. | 7464696 |
Biotin | [C@@H]12[C@H](CS[C@H]2CCCCC(O)=O)NC(=O)N1 | Primer extension by Pol III and Pol IV was observed on single DNA molecules within custom microfluidic flow cells, as previously described [12]. Briefly, primed, single-stranded DNA substrates were derived from 7.2 kb phage M13mp18 DNA (New England Biolabs) end-labeled with digoxigenin and [C@@H]12[C@H](CS[C@H]2CCCCC(O)=O)NC(=O)N1. DNAs were bound to the streptavidin-coated flow cell surface on one end, and to anti-digoxigenin-coupled 2.8 μm-diameter beads on the other. Laminar flow through the flow cell exerted a constant~3 pN force on the bead, and, by extension, uniformly throughout the tether. Conversion of entropically coiled ssDNA to extended dsDNA by a Pol at this constant force was observed as motion of the bead using dark-field microscopy. This cutoff captured~95% of events in experiments with each polymerase alone. The Pol III replisome components used in the single molecule experiments were purified as previously described: β [80]; α, δ and δ' [81]; ε and θ [82]; and τ and χψ [83]. The Pol III core αεθ and the clamp loader assembly with stoichiometry τ 3 δδ'χψ were then assembled and purified [83]. | 7464696 |
Synovial | C(CC)CNC(c1c(OCC#C)ccc(N)c1)=O | Rheumatoid arthritis (RA) is a systemic autoimmune disease. It is characterized by chronic destructive inflammation in C(CC)CNC(c1c(OCC#C)ccc(N)c1)=O joints. The prevalence of RA is about 1% in the adult European population and is three times more common in women than in men [1][2][3][4]. The aetiology of this complex disease is still poorly understood but is considered as a result of interaction between susceptibility genes and environmental factors [5]. The genetic contribution to RA has been estimated to be about 50-60% [6], with the HLA (Human leukocyte antigen) classes II molecules remaining the most powerful known genetic factor [7]. There is extensive evidence for the association between certain HLA-DRB1 alleles that contain a conserved sequence of five amino acids (Q/RK/RRAA) in the third hypervariable region of the DR 1 chain, the so-called shared epitope (SE), and RA susceptibility and severity [8][9][10]. | 16080443 |
Serine-threonine | OC(C(C(=O)O)N)C.OC(C(CO)N)=O | Genome-wide association studies (GWAS) and highdensity array Immunochip studies for single nucleotide polymorphisms (SNPs) genotyping of populations have identified over 101 RA risk loci involved among individuals of European and Asian ancestry [11][12][13][14][15]. Among these, IRAK1 (interleukin 1 receptor associated kinase) was the first X chromosome locus reported as associated with RA susceptibility and is thus of importance given the female predominance of the disease. IRAK1 is a OC(C(C(=O)O)N)C.OC(C(CO)N)=O protein kinase and an essential component of the toll/interleukin 1 receptor (TIR) signaling pathway involved in the pathogen-mediated inflammation [16]. Interestingly, the IRAK1 gene is located on the Xq28 region that harbours several SNPs that have also been associated with susceptibility to autoimmune diseases. A recent case-control study investigated numerous SNPs located on the Xq28 locus and identified rs1059703 and rs1059702, encoding for pSer532Leu and pPhe196Ser, as two IRAK1 SNPs most significantly associated with RA susceptibility in Korean families [17]. These authors also showed that the major haplotype (rs1059702 T and rs1059703 C) was associated with increased IRAK1 activity. Of note, upstream IRAK1 and within the Xq28 risk locus, Eyre et al. reported the SNP rs13397 associated with RA risk among individuals of northern European ancestry [13]. This polymorphism is located within the TMEM187 gene, which encodes a transmembrane protein of unknown function. | 16080443 |
Androgen | O(C(=O)CC)[C@@H]1[C@@]2([C@H]([C@H]3[C@@H]([C@]4(CCC(=O)C=C4CC3)C)CC2)CC1)C | O(C(=O)CC)[C@@H]1[C@@]2([C@H]([C@H]3[C@@H]([C@]4(CCC(=O)C=C4CC3)C)CC2)CC1)C receptor CCP2: Cyclic-citrullinated peptide CD40L: | 16080443 |
Tenacity | c1(cc(S(C)(=O)=O)ccc1C(C1C(=O)CCCC1=O)=O)[N+]([O-])=O | Eric Chu, the KMT candidate, provided the following narrative in the second round of cross-examination when his publicly criticized motive and action of leaving his current position as the mayor of New Taipei City to run in the presidential election was questioned by the DPP candidate Ing-wen Tsai.The confrontation at this stage characterizes the debate as one of the \essential instances of persuasive attack and defense\" (Benoit & Wells 1996).The narrative was told by Chu with a clear political aim in a political context: to answer his opponent's question | 62817287 |
Elastomers | C=C(C=C)C | Due to its ease of operation and low machine costs, FFF is already well studied for printing green parts from powder-binder formulations similar to MIM feedstock [7][8][9][10][11][12]. However, typical MIM feedstock formulations must be adapted to filament requirements such as a sufficient flexibility for spooling by adding, for instance, C=C(C=C)C [13] or amorphous polyolefins [14]. To keep changes to debinding and sintering as low as possible, the use of highly filled filaments is thus not preferable for the intended complementary green part production [6]. Screw-based extrusion, on the other hand, is suitable for this purpose, as it allows conventional MIM feedstock to be processed [5,[15][16][17][18]. Yet, machine costs are typically about ten times higher than FFF printers, since print heads are equipped with complex and expensive screw geometries [19,20]. | 245815860 |
Zeolite | [Al](O[Al]=O)=O.O=[Si]=O | [Al](O[Al]=O)=O.O=[Si]=O was successfully synthesised from ash bagasse and from rice husk ash as source of silica and applied to surfactant builder.The removal of silica from bagasse ash and from rice husk ash was influenced by NaOH concentration to obtain sodium silicate.This research aimed to synthesize [Al](O[Al]=O)=O.O=[Si]=O, determine the optimum concentration of NaOH to synthetic [Al](O[Al]=O)=O.O=[Si]=O, identify the [Al](O[Al]=O)=O.O=[Si]=O mineral type, morphology, determine cation exchange rate and detergency by using synthesized [Al](O[Al]=O)=O.O=[Si]=O as builder.Synthesis of [Al](O[Al]=O)=O.O=[Si]=O was undertaken by sol-gel method followed by hydrothermal process.The stages of this study included the production of bagasse and rice husk ashes, isolation of silicate using a variation of NaOH concentration of 1.67, 3.33, 5.00, 6.67 and 8.30 M in the form of sodium silicate.Synthesis of [Al](O[Al]=O)=O.O=[Si]=O was carried out by reacting sodium silicate and sodium aluminate using hydrothermal method.The synthesized [Al](O[Al]=O)=O.O=[Si]=Os were characterized using XRD and SEM.The results of this research indicated the types of [Al](O[Al]=O)=O.O=[Si]=O minerals formed, namely, [Al](O[Al]=O)=O.O=[Si]=O A, Na-A, Na-Y and sodalite.The morphology of the synthesized [Al](O[Al]=O)=O.O=[Si]=Os from both samples was quite homogeneous, NaOH concentration used to produce [Al](O[Al]=O)=O.O=[Si]=O from bagasse ash was 1.67 M with value of cation exchange capacity (CEC) and detergency were respectively 121.14 mek/100 gram and 92.09% while synthesis [Al](O[Al]=O)=O.O=[Si]=O from rice husk ash was generated using 8.3 M NaOH concentration with value of cation exchange capacity (CEC) and detergency were 65,71 mek / 100 gram and 94,313%, respectively. | 106285234 |
Aluminate | [OH-].[OH-].[OH-].[OH-].[Al+3] | Zeolite was successfully synthesised from ash bagasse and from rice husk ash as source of silica and applied to surfactant builder.The removal of silica from bagasse ash and from rice husk ash was influenced by NaOH concentration to obtain sodium silicate.This research aimed to synthesize zeolite, determine the optimum concentration of NaOH to synthetic zeolite, identify the zeolite mineral type, morphology, determine cation exchange rate and detergency by using synthesized zeolite as builder.Synthesis of zeolite was undertaken by sol-gel method followed by hydrothermal process.The stages of this study included the production of bagasse and rice husk ashes, isolation of silicate using a variation of NaOH concentration of 1.67, 3.33, 5.00, 6.67 and 8.30 M in the form of sodium silicate.Synthesis of zeolite was carried out by reacting sodium silicate and sodium [OH-].[OH-].[OH-].[OH-].[Al+3] using hydrothermal method.The synthesized zeolites were characterized using XRD and SEM.The results of this research indicated the types of zeolite minerals formed, namely, zeolite A, Na-A, Na-Y and sodalite.The morphology of the synthesized zeolites from both samples was quite homogeneous, NaOH concentration used to produce zeolite from bagasse ash was 1.67 M with value of cation exchange capacity (CEC) and detergency were respectively 121.14 mek/100 gram and 92.09% while synthesis zeolite from rice husk ash was generated using 8.3 M NaOH concentration with value of cation exchange capacity (CEC) and detergency were 65,71 mek / 100 gram and 94,313%, respectively. | 106285234 |
Natrium | [Na] | A b s t r a k Kata Kunci: zeolit; builder surfaktan; detergensi; sekam padi; ampas tebu Telah dilakukan sintesis zeolit dari abu ampas tebu dan dari abu sekam padi sebagai sumber silika dan diaplikasiikan untuk builder surfaktan.Pengambilan silika dari abu ampas tebu dan dari abu sekam padi dipengaruhi oleh konsentrasi NaOH untuk menperoleh [Na] silikat.Penelitian ini bertujuan untuk mensintesis zeolit, menentukan konsentrasi optimum NaOH terhadap zeolit sintetsis, jenis mineral zeolit, morfologi, menentukan nilai tukar kation dan deterjensi dengan menggunakan zeolit hasil sintesis sebagai builder.Sintesis zeolit dilakukan dengan metode sol-gel dilanjutkan proses hidrotermal.Tahapan penelitian ini meliputi pengabuan ampas tebu dan pengabuan sekam padi, pengambilan silikat menggunakan variasi konsentrasi NaOH 1,67; 3,33; 5; 6,67 dan 8,3 M sebagai [Na] silikat.Sintesis zeolit dilakukan dengan mereaksikan [Na] silikat dengan [Na] aluminat kemudian dilanjutkan proses hidrotermal.Zeolit hasil sintesis dikarakterisasi menggunakan XRD dan SEM.Hasil penelitian ini menunjukkan Jenis mineral zeolit yang terbentuk antara lain zeolit A, Na-A, Na-Y dan sodalit.Morfologi zeolit hasil sintesis dari kedua sampel cukup homogen, konsentrasi NaOH optimum adalah 1,67 M dengan nilai kapasitas tukar kation (KTK) dan detergensi berturut-turut 121,14 mek/100 gram dan 92,09 % untuk sampel abu ampas tebu sedangkan zeolit sintesis dari abu sekam padi optimum pada konsentrasi NaOH 8,3 M dengan nilai KTK dan detergensi yaitu 65,71 mek/100 gram dan 94,313%. | 106285234 |
Silikat | [O-][Si]([O-])([O-])[O-] | A b s t r a k Kata Kunci: zeolit; builder surfaktan; detergensi; sekam padi; ampas tebu Telah dilakukan sintesis zeolit dari abu ampas tebu dan dari abu sekam padi sebagai sumber silika dan diaplikasiikan untuk builder surfaktan.Pengambilan silika dari abu ampas tebu dan dari abu sekam padi dipengaruhi oleh konsentrasi NaOH untuk menperoleh natrium [O-][Si]([O-])([O-])[O-].Penelitian ini bertujuan untuk mensintesis zeolit, menentukan konsentrasi optimum NaOH terhadap zeolit sintetsis, jenis mineral zeolit, morfologi, menentukan nilai tukar kation dan deterjensi dengan menggunakan zeolit hasil sintesis sebagai builder.Sintesis zeolit dilakukan dengan metode sol-gel dilanjutkan proses hidrotermal.Tahapan penelitian ini meliputi pengabuan ampas tebu dan pengabuan sekam padi, pengambilan [O-][Si]([O-])([O-])[O-] menggunakan variasi konsentrasi NaOH 1,67; 3,33; 5; 6,67 dan 8,3 M sebagai natrium [O-][Si]([O-])([O-])[O-].Sintesis zeolit dilakukan dengan mereaksikan natrium [O-][Si]([O-])([O-])[O-] dengan natrium aluminat kemudian dilanjutkan proses hidrotermal.Zeolit hasil sintesis dikarakterisasi menggunakan XRD dan SEM.Hasil penelitian ini menunjukkan Jenis mineral zeolit yang terbentuk antara lain zeolit A, Na-A, Na-Y dan sodalit.Morfologi zeolit hasil sintesis dari kedua sampel cukup homogen, konsentrasi NaOH optimum adalah 1,67 M dengan nilai kapasitas tukar kation (KTK) dan detergensi berturut-turut 121,14 mek/100 gram dan 92,09 % untuk sampel abu ampas tebu sedangkan zeolit sintesis dari abu sekam padi optimum pada konsentrasi NaOH 8,3 M dengan nilai KTK dan detergensi yaitu 65,71 mek/100 gram dan 94,313%. | 106285234 |
Antara | C1(C=CC(OC(C)(C)C(=O)OC(C)C)=CC=1)C(C1=CC=C(C=C1)Cl)=O | A b s t r a k Kata Kunci: zeolit; builder surfaktan; detergensi; sekam padi; ampas tebu Telah dilakukan sintesis zeolit dari abu ampas tebu dan dari abu sekam padi sebagai sumber silika dan diaplikasiikan untuk builder surfaktan.Pengambilan silika dari abu ampas tebu dan dari abu sekam padi dipengaruhi oleh konsentrasi NaOH untuk menperoleh natrium silikat.Penelitian ini bertujuan untuk mensintesis zeolit, menentukan konsentrasi optimum NaOH terhadap zeolit sintetsis, jenis mineral zeolit, morfologi, menentukan nilai tukar kation dan deterjensi dengan menggunakan zeolit hasil sintesis sebagai builder.Sintesis zeolit dilakukan dengan metode sol-gel dilanjutkan proses hidrotermal.Tahapan penelitian ini meliputi pengabuan ampas tebu dan pengabuan sekam padi, pengambilan silikat menggunakan variasi konsentrasi NaOH 1,67; 3,33; 5; 6,67 dan 8,3 M sebagai natrium silikat.Sintesis zeolit dilakukan dengan mereaksikan natrium silikat dengan natrium aluminat kemudian dilanjutkan proses hidrotermal.Zeolit hasil sintesis dikarakterisasi menggunakan XRD dan SEM.Hasil penelitian ini menunjukkan Jenis mineral zeolit yang terbentuk C1(C=CC(OC(C)(C)C(=O)OC(C)C)=CC=1)C(C1=CC=C(C=C1)Cl)=O lain zeolit A, Na-A, Na-Y dan sodalit.Morfologi zeolit hasil sintesis dari kedua sampel cukup homogen, konsentrasi NaOH optimum adalah 1,67 M dengan nilai kapasitas tukar kation (KTK) dan detergensi berturut-turut 121,14 mek/100 gram dan 92,09 % untuk sampel abu ampas tebu sedangkan zeolit sintesis dari abu sekam padi optimum pada konsentrasi NaOH 8,3 M dengan nilai KTK dan detergensi yaitu 65,71 mek/100 gram dan 94,313%. | 106285234 |
Tetra sodium | C1C(C(N)=O)=CN(C2OC(C(O)C2O)COP(OP(=O)([O-])OCC2C(O)C(OP(=O)([O-])[O-])C(O2)N2C=NC3C2=NC=NC=3N)([O-])=O)C=C1.[Na+].[Na+].[Na+].[Na+] | Banyak diantara deterjen yang menggunakan builder jenis sodium tripolyphosphate (STPP) dan C1C(C(N)=O)=CN(C2OC(C(O)C2O)COP(OP(=O)([O-])OCC2C(O)C(OP(=O)([O-])[O-])C(O2)N2C=NC3C2=NC=NC=3N)([O-])=O)C=C1.[Na+].[Na+].[Na+].[Na+] pyrophosphate (TSPP), namun builder jenis tersebut dapat menyebabkan deposit fosfat dalam air dan berakibat eutrofikasi [5].Zeolit mampu menggantikan peran fosfat sebagai pembentuk builder dalam deterjen, karena zeolit dapat mencegah pembentukan garam-garam anorganik yang kurang larut dalam air [4]. | 106285234 |
Sodium pyrophosphate | O=P([O-])([O-])OP(=O)([O-])[O-].[Na+].[Na+].[Na+].[Na+] | Banyak diantara deterjen yang menggunakan builder jenis sodium tripolyphosphate (STPP) dan tetra O=P([O-])([O-])OP(=O)([O-])[O-].[Na+].[Na+].[Na+].[Na+] (TSPP), namun builder jenis tersebut dapat menyebabkan deposit fosfat dalam air dan berakibat eutrofikasi [5].Zeolit mampu menggantikan peran fosfat sebagai pembentuk builder dalam deterjen, karena zeolit dapat mencegah pembentukan garam-garam anorganik yang kurang larut dalam air [4]. | 106285234 |
Kotoran | c1c(C(F)(F)F)cccc1NC(=O)N(C)C | Bahan-bahan yang digunakan dalam penelitian ini adalah NaOH, abu ampas tebu, abu sekam padi, Al(OH)3, H2O, kain katun, Soduim Lauryl Sulfat, c1c(C(F)(F)F)cccc1NC(=O)N(C)C standart dan Na2SO4. | 106285234 |
Na2SO4 | S([O-])([O-])(=O)=O.[Na+].[Na+] | Bahan-bahan yang digunakan dalam penelitian ini adalah NaOH, abu ampas tebu, abu sekam padi, Al(OH)3, H2O, kain katun, Soduim Lauryl Sulfat, kotoran standart dan S([O-])([O-])(=O)=O.[Na+].[Na+]. | 106285234 |
Natrium | [Na] | \nPengambilan [Na] Silikat | 106285234 |
Silikat | [O-][Si]([O-])([O-])[O-] | \nPengambilan Natrium [O-][Si]([O-])([O-])[O-] | 106285234 |
Natrium | [Na] | Sebanyak 10 gram abu yang diperoleh dilarutkan ke dalam 50 mL NaOH dengan berbagai konsentrasi yaitu 1.67, 3.33, 5, 6.67 dan 8,3 M, distirer selama 2 jam 300 rpm pada suhu 80°C kemudian disaring dan filtratnya sebagai [Na] silikat. | 106285234 |
Silikat | [O-][Si]([O-])([O-])[O-] | Sebanyak 10 gram abu yang diperoleh dilarutkan ke dalam 50 mL NaOH dengan berbagai konsentrasi yaitu 1.67, 3.33, 5, 6.67 dan 8,3 M, distirer selama 2 jam 300 rpm pada suhu 80°C kemudian disaring dan filtratnya sebagai natrium [O-][Si]([O-])([O-])[O-]. | 106285234 |
Natrium | [Na] | \nPembuatan [Na] Aluminat | 106285234 |
Natrium | [Na] | Sebanyak 25 mL [Na] Silikat (5 variasi konsentrasi NaOH) dan 25 mL [Na] Aluminat dicampurkan kemudian diaduk menggunakan magnetic stirrer selama 2 jam pada 300 rpm sehingga terbentuk gel.Selanjutnya dimasukkan ke dalam autoclave dan dipanaskan pada suhu 100°C selama 340 menit dalam keadaan tertutup rapat.Hasil yang terbentuk kemudian di saring dengan kertas saring Whatmann.Padatan yang terbentuk kemudian dicuci dengan Aquades hingga pH filtrat 10-11.Selanjutnya dikeringkan pada suhu 100ºC selama 12 jam [2], dikarakterisasi dan digunakan sebagai buider. | 106285234 |
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