Astragalus taipaishanensis Y. C. Ho et S. B. Ho (A. taipaishanensis) for the first time. Their structures were elucidated by ESI-MS, HR-ESI-MS, 1D-NMR and 2D-NMR as 7,2′-dihydroxy-3′,4′-dimethoxy isoflavan (1), formononetin (2), isoliquiritigenin (3), quercetin (4), kaempferol (5), ononin (6), p-hydroxybenzoic acid (7) and vanillic acid (8). Six flavonoids (compounds 1-6) exhibited stronger antioxidant activities  than those of BHA and TBHQ and also demonstrated noticeable protective effects (particularly quercetin and kaempferol) on Escherichia coli under oxidative stress. Additionally, the chemical constituents compared with those of Astragalus membranaceus and the structure-activity relationship of the isolated compounds were both analyzed. The results clearly demonstrated that A. taipaishanensis has the potential to be selected as an alternative medicinal and food plant that can be utilized in health food products, functional tea and pharmaceutical products.Eight phenolic compounds were isolated using bio-guided isolation and purified from the roots of  Medicinal food plants are widely utilized in dietary supplements as a source of bioactive compounds that have food additive properties1Astragalus spp. has a history of being widely used in traditional medicine, coffee, tea substitutes, food and cosmetics worldwide, particularly in places such as Europe, the Middle East and Asia46A. membranaceus Bunge, which is also known as Huangqi in Chinese and Radix Astragali in Latin, belongs to the Leguminosae family. It was first recorded in Shennong Bencao Jing and its dry roots have been used in traditional Chinese medicine since 18008910Radix Astragali, such as Astragali tea and over-the-counter dietary supplement capsules, are available in U.S. health food markets12Radix Astragali have been identified and the main constituents are flavonoids, polysaccharides, saponins and amino acids14Radix Astragali exhibit noticeable antioxidant activity16The A. taipaishanensis is a species native to China that belongs to the Astragalus genus, which is only distributed in the Taibai Mountain region. In the Taibai Mountains, teas, decoctions and infusions are prepared from the roots of A. taipaishanensis as folk medicine and functional tea to enhance immunity and to improve stamina and strengthA. taipaishanensis were isolated and extracted to discover how to better utilize this plant and also attempt to gain a deeper understanding of the correlation between the health benefits of the compounds and reduced risk of diseases. The antioxidant activities of these isolated compounds were tested using several in vitro assays  along with an additional test to assess their protective effects on Escherichia coli subjected to oxidative stress by hydrogen peroxide. The results indicated that the roots of A. taipaishanensis had similar constituents and antioxidant capacities to those of A. membranaceus, indicating that it may have the potential to be developed for use in supplementary medicine and pharmaceutical products and functional food ingredients.A. taipaishanensis were analyzed  to 0.99 ± 0.004 mg/mL (WF) and 1.95 ± 0.009 mg/mL (EAF) to 6.48 ± 0.039 mg/mL (WF), respectively. In conclusion, the EAF was determined to be the fraction with the greatest antioxidant activity; therefore, later isolation and purification efforts were focused on the EAF.The TPC and TFC of the ethanolic extracts (EE) and 4 isolated fractions  of analyzed . The resA. taipaishanensis. The NMR data of the compounds are shown in 1-8 and the key HMBC correlation of compounds 1 and 2 are illustrated in Eight compounds were isolated and identified from the roots of 1 was isolated as white needle-shaped crystals. Its molecular formula was determined to be C17H18O5 on the basis of HR-ESI-MS: m/z 301.1229 [M-H]− and ESI-MS: m/z 301.15 [M-H]−. The 13C-NMR and DEPT spectra exhibited signals for two OCH3, two CH2 , six CH  and seven quaternary carbons (four O-bearing and three aromatic), respectively. The 1H-NMR spectrum of compound 1 displayed two -OCH3 signals at δ 3.78  and δ 3.79 , five aromatic proton signals at δ 6.22 , δ 6.30 , δ 6.44 , δ 6.75  and δ 6.85 , which represented the structure containing the AMX spin-coupling. When compared with published literature data, compound 1 was identified as 7, 2′-dihydroxy-3′, 4′-dimethoxy-isoflavaneCompound 2 was obtained as white needle crystal. Its molecular formula was established as C16H12O4 on the basis of HR-ESI-MS: m/z 267.0930 [M-H]− and ESI-MS: m/z 267.17 [M-H]−. 13C-NMR and DEPT spectrum showed signals for one OCH3, eight CH  and seven quaternary carbons , respectively. 1H-NMR spectrum of compound 2 exhibited one -OCH3 signal at δ 3.80 and three aromatic proton signals at δ 6.88, δ 6.95, δ 7.98 , respectively. The signal at δ 8.34  was an obvious signal of isoflavone at the H-2 position. The AA′BB′ spin-coupling system, with the signals at δ 7.00 , δ 7.52 , was ascribed to the four protons of the B-ring. After being compared with published literature values, compound 2 was identified as formononetinCompound 6 was isolated as a white amorphous powder. ESI-MS: m/z 431.32 [M+H]+. Its molecular formula was determined to be C22H22O9 based on the ESI-MS and NMR. Compared with compound 2, it had the same isoflavone skeleton. Additionally, the mass spectra and NMR spectra showed signals corresponding to a single sugar unit. The 1H-NMR spectrum of compound 6 displayed strong signals indicating anomeric protons at δ 5.16 . The sugar unit was examined by TLC analysis. Acid hydrolysis of compound 6 resulted in a product of D-glucose, which was identified by a comparison with an authentic sample. Based on the coupling constant J = 4.8 Hz greater than 4.0 Hz, the sugar configurations were identified as β-D-glucose. In addition, there was a remarkable upfield shift (Δδ 1.14) for the C-7 of compound 6 (161.91) compared to compound 2 (163.05), indicating that the sugar unit was attached to C-7. After comparison with published literature data, compound 6 was identified as ononinCompound 3 was isolated as a yellow amorphous powder. ESI-MS: m/z 255.49 [M-H]−. The 13C-NMR and DEPT spectra exhibited signals for nine CH  and six quaternary carbons , respectively. Its molecular formula was determined to be C15H12O4 based on the results from ESI-MS and NMR. The 1H-NMR spectrum of compound 3 exhibited three aromatic proton signals at δ 6.30 , δ 6.43 , and δ 7.99 , and it was ascribed to three protons of the A-ring of flavone. The signal at δ 7.63  and δ 7.81 were obvious signals of conjugate protons; moreover, there was an AA′BB′ spin-coupling system with signals at δ 6.87 , δ 7.65, which was attributed to the four protons of the B-ring. When compared with published literature data, compound 3 was identified as isoliquiritigeninCompound 4 was isolated as a yellow amorous powder. ESI-MS: m/z 301.29 [M-H]−. The 13C-NMR and DEPT spectra exhibited signals for five CH  and ten quaternary carbons . Its molecular formula was determined to be C15H10O6 based on the results of ESI-MS and NMR. In the 1H-NMR spectrum, an ABX spin-coupling system was ascribed to the three protons of the B-ring of the flavone, with signals at δ 6.89 , δ 7.54 , and δ 7.65 , and with respect to published literature data, compound 4 was identified as quercetinCompound 5 was isolated as a yellow amorous powder. ESI-MS: m/z 284.96 [M-H]−. The 13C-NMR and DEPT spectra displayed signals for six CH  and nine quaternary carbons . Its molecular formula was determined to be C15H10O5 based on the ESI-MS and NMR spectra. Compound 5 was found to have the same skeleton as compound 4. In the 1H-NMR spectrum, the AA′BB′ spin-coupling system with signals at δ 6.92  and δ 8.04  was ascribed to the four protons of the B-ring. The signals at δ 6.19  and δ 6.45  were meta-substituted protons of the A-ring. When compared with published literature data, compound 5 was identified as kaempferolCompound 7 and compound 8 were identified by TLC analysis and a comparison with an authentic sample. Compound 7 was identified as p-hydroxy benzoic acid, and compound 8 was identified as vanillic acidCompound 1–8 isolated from A. taipaishanensis; furthermore, the 2D-NMR data of compounds 1 and 2 were presented for the first time in this study.To the best of our knowledge, this is the first report of the characterization of phenolic compounds 1–6 were tested for antioxidant by using four in vitro antioxidant assays and protective effect on E. coli under peroxide stress. The results showed in the 4 and 5 had comparative higher antioxidant capacity with ABTS values over 30 mmol trolox/g. FRAP assay, compounds 4, 5 and 1 had higher antioxidant activity compared to BHA (5.83 ± 0.08 mmol trolox/g) and TBHQ (6.57 ± 0.13 mmol trolox/g), and the values were 14.18 ± 0.13, 14.13 ± 0.16 and 10.77 ± 0.11 mmol trolox/g, respectively. The results of DPPH assay demonstrated that all the tested compounds had stronger DPPH radical-scavenging ability than the standards (BHA and TBHQ). Compounds 4, 5, 1 and 3 in particular had relatively higher antioxidant activity, in which the IC50 values were lower than 1 μM. The lipid peroxidation assay was carried out using egg yolk as a lipid-rich medium. For this assay, all the tested compounds exhibited a particular capacity for lipid peroxidation inhibition. In particular, compounds 4, 5 and 1 had comparatively higher lipid peroxidation potential with IC50 values 1.65 ± 0.08, 1.30 ± 0.09 and 1.87 ± 0.07 μM, respectively, all of which were lower than 2 μM.Phenolic compounds possess biologically beneficial activities and are frequently utilized as antioxidant, antiallergic and antidiabetic reagentsE. coli growth under peroxide stress. We evaluated and compared the specific growth rate (μ) of E. coli cells by pretreating them with the 6 compounds before subjecting them to oxidative stress damage caused by H2O2 for 30 min. The absorbance (OD600) and μ values are displayed in 600 were indicative of a higher antioxidant potential for the compounds. The results demonstrated that all the compounds had the ability to increase the cell growth rate under oxidative stress by 3.86- to 5.56-fold compared to untreated cells subjected to oxidative stress for 30 min. The compound with the highest activity was compound 4, which resulted in a growth rate of 5.56, followed by compound 3 and compound 1, which resulted in growth rates of 4.14 and 4.09, respectively.Six of the isolated compounds were tested on A. membranaceus and A. membranaceus var. mongholicus are mainly grown in North, Northeast and Northwest China and have been widely used for 2000 yearsA. membranaceus30A. membranaceusA. membranaceus. Due to its known multiple health benefits, the natural sources of A. membranaceus are diminishing yearlyA. taipaishanensis for the first time, examining and evaluating their bioactivities by in vitro radical scavenging, ferric reducing power and lipid peroxidation in addition to assessing their protective ability on E. coli cells subjected to oxidative stress damage caused by H2O2.1), formononetin (2), isoliquiritigenin (3), quercetin (4), kaempferol (5), ononin (6), p-hydroxy benzoic acid (7) and vanillic acid (8) from the roots of A. taipaishanensis. When compared with published literature, it was discovered that these 8 compounds isolated from A. taipaishanensis were all present in A. membranaceus, particularly isoliquiritigenin, formononetin, ononin, kaempferol, quercetin, which are the main beneficial flavonoids in A. membranaceus; therefore, it is possible to infer that A. taipaishanensis would contain similar flavonoid constituents to those of A. membranaceus. This could also explain the popular use the roots of A. taipaishanensis as folk medicine and functional tea to enhance the immune system and improve stamina and strength.We isolated 7, 2′-dihydroxy-3′,4′-dimethoxy isoflavan  and kaempferol (5), always had the highest antioxidant activities. In contrast, 7, 2′-dihydroxy-3′,4′-dimethoxy isoflavan (1) had a slightly lower antioxidant activity than flavonol followed by chalcone  and the isoflavone , and isoflavone glucoside  had the lowest antioxidant capacities. These results were consistent with previous reports stating that the number and position of hydroxyl groups and the different flavonoid skeletons would largely influence the radical scavenging ability. Previous reports stated that the 3-OH group would significantly increase the radical scavenging power34α, β-double bond in isoliquiritigenin made its anti-radical potential evident36By comparing the results of four A. taipaishanensis. We isolated 6 flavonoid compounds and 2 phenolic compounds from A. taipaishanensis, all of which were isolated for the first time from this species. Additionally, we found that the main chemical components and the bioactivity of A. taipaishanensis were similar to those found in A. membranaceus, which is recorded in the pharmacopoeia as a medicinal and food plant. The antioxidant activities and structure-activity relationships of these isolated compounds were also evaluated, and the results revealed that all the tested compounds exhibited noticeable antioxidant activity compared to BHA and TBHQ. These findings demonstrate the potential of A. taipaishanensis to serve as a valuable natural resource in the medicinal and food industry and its ability to be used as a possible alternative in response to the diminishing supply of A. membranaceus.In conclusion, the present study, to the best of our knowledge, was the first time a comprehensive study was carried out on the phytochemicals and antioxidant capacity of the 18 column . HR-ESI-MS was performed on 1260 HPLC combined with 6250 ESI-Q-TOF MS . HPLC separation was performed on an Agilent 1260 HPLC with a Zobax Eclipse Plus C18 column . 1H-and 13C-spectra were measured with a Bruker AVANCE III spectrometer (500 MHz). Thin layer silica gel and column gel were both purchased from Qingdao Ocean Company (China). Sephadex LH-20 was purchased from GE Healthcare Bioscience AB (Sweden).ESI-MS was performed on an Agilent 1200 HPLC with the 6130 SQD system and a Waters XBridge C3·6H2O), potassium persulfate ; tetrahydrofuran (THF) ; quercetin, formononetin, glucose ; yeast extract, tryptone . All the chemicals used including the solvents were of analytical grade.The following chemicals and reagents were used: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulphonic acid) di-ammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ; thiobarbituric acid (TBA) ; sodium borohydride (NaBH4), vanillin, aluminum chloride, acetic acid, hydrochloric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium carbonate, sodium acetate, ferric trichloride hexahydrate  were extracted five times each with 95% ethanol (10 L) for 24 h at room temperature. The ethanol extracts  were evaporated in vacuum at 45 °C and dried to solid at 35 °C. The EE (420 g) was further partitioned with petroleum ether-water, ethyl acetate-water and n-BuOH-water to obtain the 4 fractions: petroleum ether fraction , ethyl acetate fraction , n-butanol fraction  and water fraction , respectively. The yield of EE and the four fractions was 16.27%, 5.71%, 4.29%, 9.52% and 80.48%, respectively.The dried roots of 1 (110 mg). Fr. 3  was subjected to a silica gel column  using a petroleum ether/acetone gradient elution (100:0 to 0:100) to yield 4 sub-fractions (Fr. 3.1–3.4). Fr. 3.2  was further isolated by using a silica gel column  and an ethyl acetate/benzene gradient elution (100:0 to 0:100) to yield 3 fractions (Fr. 3.2.1–3.2.3). Fr. 3.2.2  was further purified by HPLC  to obtain compound 2 (35 mg). Fr. 3.4  was subjected to a silica gel column  using a petroleum ether/acetone gradient elution (100:0 to 0:100) to yield 3 sub-fractions (Fr. 3.4.1–3.4.3). Fr. 3.4.2  was subjected to a silica gel column  to further isolation by eluting with dichloromethane/benzene (100:0 to 0:100) and yield 3 fractions (Fr. 3.4.2.1-3.4.2.3). Fr. 3.4.2.2  was purified by using a silica gel column  and a petroleum ether/ethyl acetate gradient elution (100:0 to 0:100) to yield 2 fractions (Fr. 3.4.2.2.1–3.4.2.2.2). Fr. 3.4.2.2.1 was further purified by HPLC  to obtain compound 3 (25 mg). Compound 7 (22 mg) was purified by HPLC  from Fr. 3.4.2.2.2. Compound 8 (23 mg) was purified by HPLC  from Fr. 3.4.2.3. Fr. 4  was subjected to a silica gel column  using a petroleum ether/acetone gradient elution (100:0 to 0:100) to yield 4 sub-fractions (Fr. 4.1–4.4). Fr. 4.2  was subjected to a silica gel column  using a dichloromethane/acetone gradient elution (100:0 to 0:100) to yield 3 fractions (Fr. 4.2.1–4.2.3). Fr. 4.2.2  was applied to a Sephadex LH-20 column (1000 × 15 mm) and eluted with MeOH to obtain compound 4 (21 mg). Fr. 4.3  was subjected to a silica gel column  using a dichloromethane/acetone gradient elution (100:0 to 0:100) to obtain 2 fractions (Fr. 4.3.1–4.3.2). Fr. 4.3.1  was further purified by HPLC  to obtain compound 5 (24 mg). Fr. 5 (2.3 g) was subjected to a silica gel column  using a petroleum ether/acetone gradient elution (100:0 to 0:100) to yield 4 fractions (Fr. 5.1–5.4). Fr. 5.4  was subjected to a silica gel column  using a dichloromethane/acetone gradient elution (100:0 to 0:100) to yield 2 fractions (Fr. 5.4.1–5.4.2). Fr. 5.4.2  was applied to a Sephadex LH-20 column (1000 × 15 mm) and eluted with MeOH to obtain Fr. 5.4.2.1 (250 mg). Compound 6 (36 mg) was purified by HPLC  from Fr. 5.4.2.1.The EAF (18 g) was purified using a silica gel column  and successively eluted with a petroleum ether/ethyl acetate gradient elution (90:10 to 0:100) to yield 5 fractions (Fr. 1-Fr. 5). Fr. 1  was subjected to a silica gel column  using a petroleum ether/ethyl acetate gradient elution (90:10 to 0:100) to yield 3 fractions (Fr. 1.1–1.3). Fr. 1.3  was subjected to a silica gel column  using a dichloromethane/benzene gradient elution (100:0 to 0:100) to obtain 3 fractions (Fr. 1.3.1–1.3.3). Fr. 1.3.2  was purified by silica gel column  and eluted with a petroleum ether/ethyl acetate gradient elution (90:10 to 0:100) to obtain compound 6 (5.0 mg) was dissolved in 5.0 mL of 2 N HCl, heated at 90 °C for 2 h and then partitioned between ethyl acetate and water. A glycon formononetin was recovered from the ethyl acetate layer and identified by direct comparison with an authentic sample. The water layers were identified by comparison with an authentic sample using TLC analysis6 were identified as glucose.Compound The total phenolic content (TPC) was determined using a modified version of the Folin-Ciocalteau colorimetric methodThe total flavonoid content (TFC) was determined using a sodium borohydride/chloranil-based (SBC) assayThe radical scavenging activity of extracts and compounds were measured by a slightly modified version of the method described by Brand-WilliamsADPPH is the absorbance of the DPPH solution, Ablank is the absorbance of methanol instead of DPPH, As-DPPH is the absorbance of the DPPH solution in the presence of the sample, and As-blank is the absorbance of methanol in the presence of the sample.where The ABTS assay was performed as previously described in published literature with some modificationsThe FRAP assay used was a modified version of that reported by Benzie and StrainIn this assay, a modified thiobarbituric acid reactive species (TBARS) assay was used to measure the amount of lipid peroxide formed. The egg yolk homogenates were used as a lipid-rich medium4450 value was calculated from the regression equation obtained from plotting the sample concentration and rate of inhibition.where C is the absorbance of fully peroxidized control and T is the absorbance of test sample. BHA and TBHQ were used as reference standards. The ICE. coli (ATCC No. 25922) was used to determine the antioxidant activity of compounds in vivo based on a modified version of the method described by Smirnova46600 of 0.25 ± 0.004. Then, 100 μL of the compounds (0.1 mM), a 1-mL cell suspension and 8.9 mL of LB medium were mixed and incubated at 37 °C with shaking at 180 r/min for 1.5 h. After the 1.5 h incubation, 6.5 mM of hydrogen peroxide was added and the absorbance at 600 nm was then measured immediately. The sample was then incubated at 37 °C with shaking at 180 r/min for an additional 30 min before the absorbance was measured again. The OD600 is used to indicate the growth of E. coli, with a higher value of the OD600 indicating a higher growth rate of E. coli. The isolated compounds have the ability to reduce and eliminate the oxidation of H2O2 and protect the growth of E. coli; therefore, the higher antioxidant ability of the compounds indicates that the specific growth rate will be higher.The microbial test system used in the manuscript was an effective method to evaluate the antioxidant properties of medicinal plant extracts and polyphenols. This method is easier in terms of experimental operation, lower in cost compared to cellular antioxidant activity assays and more biological relevant than the popular chemistry antioxidant activity measures. In this study, Specific growth rate was calculated according to the following equation:0 and N are the optical density at time zero and t, respectively.where μ is the specific growth rate and NAll results are expressed as the mean ± standard deviation (SD). SPSS  software was used to determine significant differences via one-way ANOVA followed by Duncan’s test; values < 0.05 were considered to be significant.How to cite this article: Pu, W. et al. Structural Characterization and Evaluation of the Antioxidant Activity of Phenolic Compounds from Astragalus taipaishanensis and Their Structure-Activity Relationship. Sci. Rep.5, 13914; doi: 10.1038/srep13914 (2015).


The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S , 11S acidic  and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride. Soymilk is a colloidal solution containing 3.6% protein and is prepared by soaking and grinding soybeans in water, followed by filtering and heating. β-Conglycinin (7S) and glycinin 11S) are the two major soymilk proteins, representing approximately 40% and 30% of the total protein, respectively1S are thIn addition to protein, soymilk is an excellent source of isoflavones. These isoflavones have received considerable attention due to their biological activity, such as lowering lipid and blood glucose levels, displaying antiatherosclerotic and antioxidation properties, and preventing hormone-dependent breast and prostate cancers5Soymilk can be transformed into soybean curd, also known as tofu. Tofu is a gel-like soybean food, formed by the addition of coagulants, resulting in the formation of a protein matrixAlthough the soymilk proteins were coagulated by calcium chloride, it is unclear how the isoflavones were precipitated into the tofu during the production process. To investigate the effect of calcium chloride on the coagulation of 7S, 11S and isoflavones in soymilk, SDS-PAGE, 2-DE and HPLC were conducted. Therefore, the objective of this study was to investigate the calcium chloride-induced coagulation of 7S, 11S and isoflavones in soymilk.P < 0.05). The concentration of calcium in soymilk is very low (<0.02 mM) and that adding 25 mM calcium chloride to soymilk can precipitate soymilk proteins2+-induced protein aggregation is thought to arise from three effects: 1) electrostatic shielding, 2) ion-specific hydrophobic interaction, and 3) crosslinking of adjacent anionic molecules through the formation of protein-Ca2+-protein bridges. The binding of Ca2+ to soy proteins is an endothermic process (ΔH > 0) based on binding titration curves of soy protein dispersions with calcium chlorideSoymilk samples were incubated with varying concentrations of calcium chloride  for 1 h. The amount of total protein in the SSF and SPF was determined. As shown in P < 0.05), the protein bands corresponding to the these proteins decreased to 1.8 ± 0.1, 0.9 ± 0.1, 1.1 ± 0.3, 1.6 ± 0.2, 2.1 ± 0.1 and 1.6 ± 0.1%, respectively  were analyzed by SDS-PAGE . The intectively . These pectively . The preI)  . These pSoymilk samples treated with varying amounts of calcium chloride  were also analyzed by 2-DE gels . When noThe isoflavones extracted from soymilk and 7S and 11S proteins were analyzed by HPLC . IsoflavThe following sections will focus on the spectral data of the identified daidzin, glycitin, genistin, daidzein and genistein obtained from ESI-MS, FT-IR and UV.m/z 417.1181 [M+H]+ . IR νmax: 3411, 1634, 1515, 1446, 1249. UV (acetone: water: acetic acid = 70:29.5:0.5) λmax nm: 232, 260. Compound 1 was identified as daidzin by direct comparison with commercial daidzin . Based on the data described above and the verified referenceCompound 1 was a white powder , ESI-MS:m/z 447.1286 [M+H]+ . IR νmax: 3342, 1635, 1518, 1498, 1469, 1432, 1276, 1217, 1087, 1040. UV (acetone: water: acetic acid = 70:29.5:0.5) λmax nm: 248, 220. Compound 2 was identified as glycitin by direct comparison with commercial glycitin . Based on the data described above and the verified referenceCompound 2 was a white powder , ESI-MS:m/z 433.1130 [M+H]+ . IR νmax: 3440, 1659, 1621, 1580, 1520. UV (acetone: water: acetic acid = 70:29.5:0.5) λmax nm: 228, 260. Compound 3 was identified as genistin by direct comparison with commercial genistin . Based on the data described above and the verified referenceCompound 3 was a white powder , ESI-MS:m/z 255.0651 [M+H]+ . IR νmax: 3221, 1632, 1593, 1518. UV (acetone: water: acetic acid = 70:29.5:0.5) λmax nm: 224, 248. Compound 4 was identified as daidzein by direct comparison with commercial daidzein . Based on the data described above and the verified referenceCompound 4 was a white powder , ESI-MS:m/z 271.0601 [M+H]+ . IR νmax: 3440, 3069, 2922, 1655, 1622, 1178, 810, 785. UV (acetone: water: acetic acid = 70:29.5:0.5) λmax nm: 232, 260. Compound 5 was identified as genistein by direct comparison with commercial genistein . Based on the data described above and the verified referenceCompound 5 was a white powder , ESI-MS:P < 0.05). However, only a portion of daidzin, gycitin and genistin in the SSF decreased with 5 mM calcium chloride. The amounts of daidzein, genistein, daidzin, gycitin and genistin in the SSF decreased to 39.4 ± 1.6, 11.8 ± 7.0, 91.4 ± 4.0, 75.8 ± 1.2 and 80.1 ± 6.4%, respectively, during that period. These isoflavones, including daidzein and genistein, and a portion of daidzin, gycitin and genistin also appeared in the SPF , glycinin (11S), and isoflavones in soymilk. The results obtained from SDS-PAGE analysis, two-dimensional electrophoresis, and HPLC clearly demonstrate that the addition of 5 mM calcium chloride induced the coagulation of most of the 7S , 11S acidic , and 11S basic (B1a) proteins was well as isoflavones, including daidzein and genistein. Moreover, we provide evidence that isoflavones (including daidzein and genistein) bonded to 7S and 11S to form 7S-isoflavone complex and 11S-isoflavone complex in soymilk. These complexes were then precipitated into SPF following the addition of 5 mM calcium chloride. Our findings provide important information related to the effects of calcium chloride on the coagulation of 7S, 11S, and isoflavones, and provide a valuable reference for future research into the production of tofu from soymilk.Glycine max (L.) Merr., 100 g] were washed and soaked in distilled water at 25 °C for 12 h. Hydrated seeds were drained and ground into homogenates in 1 L of distilled water. The raw soymilk was filtered through a cotton filter and heated in a 90 °C water bath for 1 h. The soymilk was collected and stored at 4 °C. To investigate the effects of calcium chloride on the coagulation of soymilk proteins, soymilk was centrifuged at 12,000 × g for 20 min at 4 °C using a centrifugal separator to remove lipids. Varying concentrations of calcium chloride  were added to 1 mL of soymilk, followed by incubation at 30 °C for 1 h. Following incubation, the soymilk samples were fractionated into the soymilk supernatant fraction (SSF) and the soymilk pellet fraction (SPF) by centrifugation for 20 min . The SSF samples (1 mL) were collected, and the SPF samples were re-dissolved in an equal volume (1 mL) of a lysis solution containing 7 M urea, 2 M thiourea, and 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate prior to use.Soybeans -1-propanesulfonate, 4 mM Tris-base, 2% IPG ampholyte, 65 mM 1,4-dithioerythritol (DTE) and 0.0002% bromophenol blue prior to use. Isoelectric focusing of the strips was performed using the IPGphor 3 IEF system  at 20 °C at 6000 V, for a total of 60 kVh. The strips were equilibrated for 15 min in an equilibration solution  and added to the top of a vertical 12.5% SDS-PAGE gel with 0.5% agarose. The second electrophoresis step was performed using the Protean II xi Cell system  at 10 mA per gel for 1 h and 45 mA per gel for 5 h until the bromophenol blue reached the bottom of the gel. After electrophoresis, the gels were immersed in 10% methanol and 7% acetic acid for 30 min and stained in 350 mL of SyproTM; Micromass, UK). Peptide mass fingerprint data from MALDI-Q-TOF analyses were used to identify protein candidates in the Swiss-Prot protein database using the Mascot (http://www.matrixscience.com) search program to identify proteins. The search parameters allowed for methionine oxidation, cysteine carbamidomethylation, one missed cleavage site and a peptide mass tolerance of 0.25 Da. The product ion spectra generated by Q-TOF MS/MS were compared to the Swiss-Prot protein database, and an exact match was found using the Mascot search program.Fourteen protein spots on the 2-DE were excised and destained in a solution containing 250 μL acetonitrile/50 mM ammonium bicarbonate (1:1). The gels were dried using a centrifugal vacuum concentrator. The cysteine residues in the samples were reduced and alkylated using DTE and iodoacetamide, respectively. The gels were rehydrated in a trypsin solution (12.5 ng/mL) for tryptic digestion and incubated at 37 °C for 16 h. Peptide fragments were extracted with an equal volume of 100% acetonitrile and 2% trifluoroacetic acid, and sonicated in a water bath for 10 min. The extracted peptides were concentrated in a vacuum centrifuge. For matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis, the extracted peptides were mixed 1:1 with a matrix solution  and spotted onto a 96-well format MALDI sample stage. Data were directly obtained using a quadrupole time-of-flight (Q-TOF) Ultima MALDI instrument . The statistical significance between the treatments was determined by one-way ANOVA followed by Duncan’s multiple range test. Three determinations for each treatment were performed, and the significance level was set at How to cite this article: Hsiao, Y.-H. et al. Coagulation of β-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk. Sci. Rep.5, 13018; doi: 10.1038/srep13018 (2015).


This strategy, however, may induce hypercapnic acidosis, promote derecruitment and, in some individuals, induce alveolar overdistention despite the use of low Vt. Extracorporeal CO2 removal in ARDS during ultraprotective ventilation in terms of lung mechanics and gas exchange.To evaluate the effect of extracorporeal COultraprotective ventilation (i.e. Vt 4 ml/kg PBW) was implemented by means of an extracorporeal CO2 removal system . Anticoagulation with unfractionated heparin to reach an aPTT target range of 1.5-2 was used. We compared baseline ventilation with ultraprotective ventilation (combining Vt of 4 ml/kg PBW and Hemolung®), in terms of lung mechanics and gas exchange. We collected arterial blood gases, respiratory and hemodynamic variables, and mixed expired gases at baseline and after 60 minutes of stabilization at ultraprotective ventilation. Statistical analysis: 2-tailed Student's t-test. Statistical significance p < 0.05.We studied 9 ARDS patients, in whom ® allowed a CO2 removal rate of 84 ± 9 mL/min, with blood flow 447 ± 35 mL/min, at constant sweep gas flow (10 L/min of O2) and pump speed (1400 RPM). Unfractionated heparin dose was 200 ± 78 mg/day and aPTT was 1,56 ± 0.18. During catheter insertion a bolus of 0.6 ± 0.2 mg/kg mg was administered. Hemolung® total days were 5.3 ± 6.2 (range 1 to 22). No significant haemorrhage or hemolysis needing transfusion, device malfunction, insertion and/or withdrawal complications occurred. We report a significant reduction in minute ventilation and alveolar minute ventilation , dead space (68%), and driving pressure (69%), without significant changes in arterial blood gases when ultraprotective strategy was implemented, as compared to baseline . Age was 61 ± 14 years, SAPS II at admission 48 ± 28 and ICU mortality 22% (2/9). Seven of these patients were treated with prone positioning during mechanical ventilation. Cannulation was done via femoral vein in all patients, using “ad hoc” 15.5 Fr catheters. Hemolung® system allows ultraprotective ventilation, while maintaining adequate arterial blood gases and significantly decreasing the intensity of ventilator assistance. The technique appears to be useful and safe.HemolungMaterial for the study was kindly provided by ALung, Pittsburgh, USA.


The obtained nanoparticles (HATXP) were biocompatible and enzymatic biodegradable due to their hydrophilic hyaluronic acid (HA) shell and hydrophobic Taxol core, and exhibited specific targeting internalization into cancer cells via HA receptor mediated endocytosis effects. The cytotoxicity experiments showed that the HATXP exhibited similar anticancer activities to, but much lower side effects than commercial anticancer drug Taxol. The present work would provide a platform for targeted paclitaxel drug delivery and a general protocol for the design of advanced multifunctional nanoscale biomaterials for targeted drug/gene delivery.The construction of synthetic straightforward, biocompatible and biodegradable targeted drug delivery system with fluorescent tracking abilities, high anticancer activities and low side effects is still a challenge in the field of biochemistry and material chemistry. In this work, we constructed targeted paclitaxel (Taxol) delivery nanoparticles composed of permethyl- However, the complicated synthetic routes, multi-stepped modification and separation, insufficient targeting capability, undefined mechanism of drug release and cytotoxicity of the drug carriers would not facilitate their benefits in anticancer therapy and therefore impede their practical applications in clinic.The construction of novel diagnosis and drug-therapy systems against cancer nowadays becomes research hotspots in the fields of biochemistry and material chemistry12457810111314On the other hand, the early diagnosis of cancer plays an important role in prevention, early detection and early treatment of cancer and the early find of cancer might promote the survival rate of the patients suffered from cancer. Therefore, the development of novel and sensitive diagnostic reagents toward cancer also draw attention of the interest of medical researchers. Therefore a large amount of fluorescent16171820212324β-cyclodextrin (permethyl-β-CD) and porphyrin derivates had been reported in numerous supramolecular architectures293032in vitro and in vivo without obvious side effects. For instance, Chen et al.β-cholanic acid/ZnII-dipicolylamine tethered HA and pH-dependent CaP shell, and the obtained nanoparticle could strongly bind anticancer drug doxorubicin and multidrug resistance 1 gene target siRNA, and then after associating with the CD44 receptor over-expressed cancer cells, the effective drug/siRNA release and the targeted cytotoxicity toward drug resistant cancer cells could be realized in vitro and in vivo due to the suppression of target gene expression. Furthermore, Hahn et al.et al.in vitro and in vivo. However, the relative complicated synthetic routes would not facilitate the practical application of the nanoparticles in clinic. Previously our groupβ-cyclodextrin modified HA and adamantane modified cisplatin prodrug via host-guest and amphiphilic interactions, and the obtained nanoparticle could specifically target HA-receptor positive cancer cells, and exhibit better anticancer activities and lower side effects than commercial chemotherapy drug cisplatin in vitro and in vivo.Importantly, the convenient construction and controllable drug release of targeted drug delivery system could be probably realized by supramolecular chemistry and amphiphilic interactions without complicated modification process2627β-CD modified HA (HApCD) and porphyrin modified paclitaxel (PorTaxol) through amide condensation reaction and esterification reaction, respectively. Taking advantage of the ultra-strong host-guest interaction between permethyl-β-CD and porphyrin part, we got a supramolecular complex with amphiphilicity, and this amphiphilic complex could self-assemble to form nanoparticles HATXP which had a hydrophilic HA shell that could target HA-receptor over-expressed on the surface of cancer cells, and a hydrophobic PorTaxol prodrug core that possessed fluorescent labeled function  and characterized by 1H NMR spectroscopy and gel permeation chromatography (GPC). As shown in β-CD appeared around the chemical shift of 5 ppm, and the signals assigned to H2-H6 protons of permethyl-β-CD’s and HA’s protons appeared around 3–4 ppm, which both indicated the successful grafting of permethyl-β-CD onto the skeleton of HA molecule. After analyzing the integral values of the peak area of permethyl-β-CD’s and HA’s protons, we could calculate that there is one permethyl-β-CD modified on every 7.37 polysaccharide units of HA, and this degree of substitution (DS) may not impede the targeting properties of HA backbone toward CD44 receptors overexpressing on the surface of malignant cancer cells which require at least six repeating units to form a targeting section5, which is very consistent with the weight-average molecular weight , see 5 determined by GPC. The increasing molecular weight of HApCD compared with HA (Mw = 1.9 × 105) further confirms the successful modification of permethyl-β-CD onto HA molecule. The characterization data of HApCD from 1H NMR and GPC were summarized in α in Taxol showed a large shift from 4.96 ppm to 5.74 ppm after the modification of Taxol with porphyrin, which revealed that the carboxyl group of porphyrin molecule reacted with the hydroxide group of Taxol on C-α, and then lead to the changes in the chemical environment of H-α.The synthetic routes of HApCD and the porphyrin modified paclitaxel (PorTaxol) were shown in β-CD cavity in HApCD. As shown in β-CD, indicating that the porphyrin backbone in PorTaxol was included into the permethyl-β-CD cavityβ-CD cavity (KS) between PorTaxol and permethyl-β-CD was calculated as (1.04 ± 0.25) × 106 M−1, according to the results in the UV/Vis spectral titration by analyzing the sequential changes of the absorption intensity of PorTaxol at varying the concentrations of permethyl-β-CD.The UV/Vis spectral titration was carried out to investigate the association between PorTaxol and the permethyl-D cavity . Therefoβ-CD cavity in HApCD, a supramolecular nanoparticle (HATXP) containing hyaluronic acid as a targeting unit and Taxols as bioactive units was successfully constructed by simply mixing the HApCD and PorTaxol in aqueous solution. Then the obtained mixture was dialyzed against an excess amount of deionized water for 2 h to remove any unbounded PorTaxol. Then the drug loading ratio (the weight ratio of loaded drug to carrier) and drug encapsulation ratio of PorTaxol were measured as 31% and 85%, respectively, through the photometric standard curve of PorTaxol at λ = 515 nm , two permethyl-β-CDs (2.0 nm), and two Taxol molecules (2.0 nm)ca. 171 nm with a narrow distribution (ca. −29 mV (ca. −34 mV) or HA (−15 mV) (in vivoThen the size and morphology of HATXP were measured by atomic force microscopy (AFM), high-resolution transmission electron microscopy (HR-TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), and zeta potential experiments. As shown in ribution . The zet. −29 mV , which w(−15 mV)  owing toβ-CD with porphyrin, HApCD and PorTaxol could form an amphiphilic complex, which further assembled to nanoparticles possessing the hydrophobic PorTaxol core and the hydrophilic HA shell with great biocompatibility and targeting capability toward cancer cells.The biocompatibility of PorTaxol and HATXP were measured comparatively in PBS. As shown in ca. 80%, indicating that a majority of HATXP was biodegraded by hyaluronidase  and NIH3T3 mouse embryonic fibroblast cell line (HA-receptor negative) as model cells. As shown in 41μM and 2.89 μM, respectively, which would further prove our conclusion that HATXP showed comparable anticancer activity to commercial Taxol.The anticancer effect of HATXP nanoparticles was evaluated by the cytotoxicity experiments. As shown in It is noteworthy that, although HATXP showed a bit lower anticancer activity than Taxol (relative cellular viability 26% by HATXP vs 17% by Taxol towards SKOV-3 cells), its toxicity towards normal cells (relative cellular viability 84% by HATXP vs 10% by Taxol towards NIH3T3 cells) was very lower than that of Taxol . The resIn conclusion, a targeted nanoparticle with hydrophobic PorTaxol core and hydrophilic HA shell was constructed through supramolecular and amphiphilic interactions. The nanoparticles could specifically recognize malignant cancer cells through HA-HA receptor interactions and induce the apoptosis of cancer cells without obvious side effects toward normal cells. On the other hand, taking advantage of the fluorescent porphyrin part, the location of the nanoparticles in cells could be explicit. Therefore, these nanoparticles could act as an integrative diagnosis-treatment platform, and the potential applications of nanoparticles in photodynamic therapy are still in process.β-CD was recrystallized twice from water and dried in vacuo at 90 °C for 24 h prior to use. Sodium hyaluronate , paclitaxel (Taxol), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysulfosuccinimide (NHSS), N,N′-dicyclohexyl-carbodiimide (DCC), 4-dimethylaminopyridine (DMAP), and hyaluronidase were purchased from commercial sources and used as received. Mono-6-deoxyl-6-ethylenediamino-permethyl- β-CD2 for 1 day and then distilled at room pressure prior to use. Column chromatography was performed on 200–300 mesh silica gel.All chemicals were reagent-grade unless noted otherwise. 2 F20 microscope instrument operated at 200 kV. The samples were prepared by placing a drop of solution (0.1 mg/mL) onto a carbon-coated copper grid. The samples for SEM measurements were prepared by dropping each sample solution onto a coverslip followed by evaporation of the solvent at room temperature. SEM images were obtained on a Shimadzu SS-550 scanning electron microscope. The sample solutions for DLS experiments were prepared by filtering each solution through a 450 nm syringe-driven filter (JET BIOFIL) into a clean scintillation vial. The samples were examined on a NanoBrook 173Plus at λ = 636 nm at 25 °C. All DLS measurements were performed at the scattering angle of 90°. The zeta potential was recorded on a NanoBrook 173Plus at 25 °C. GPC measurements were employed to examine the weight-averaged molecular weights (Mw) of polymers on a four detection size exclusion chromatograph (Four-SEC) containing a Waters 1525 separation module connected with a Viscotek M302 four detector array, a combination of refractive index, light scattering , viscosity detector, and UV/Vis detector. Two mixed bed SEC columns  were used. Poly(ethylene oxide) was used as calibration standard and 0.1 M phosphate buffer (pH = 7.2) was used as a mobile phase at a flow rate of 1.0 mL min−1 at an operating temperature of 25 °C. The fluorescence confocal images were carried out on Leica TCS SP8 fluorescence microscope .NMR spectra were recorded on a Bruker AV400 instrument. UV/Vis spectra were recorded in a conventional quartz cell (light path 10 mm) by using a Shimadzu UV-2401PC spectrophotometer equipped with a Thermo HAAKE-SC100 temperature controller to keep the temperature at 25 °C. For the AFM measurements, a sample solution (0.1 mg/mL) was dropped onto newly clipped mica and air-dried, and the residue obtained was examined in tapping mode in the air under ambient conditions using a Veeco Nano IIIa Multimode AFM instrument. High-resolution transmission electron microscope (HR-TEM) images were obtained on a Tecnai GN-hydroxysulfosuccinimide sodium salt (NHSS)  were added to a solution of sodium hyaluronate  in phosphate buffer saline  and the mixture was stirred at 25 °C for 30 min, then mono-6-deoxyl-6-ethylenediamino- permethyl-β-CD  in PBS (10 mL) was added. The reaction mixture was stirred at room temperature for 24 h. The resulting solution was dialyzed against excess amount of deionized water for 5 days. After being freeze-dried, the product HApCD was obtained as a white powder. 1H NMR : δ = 1.87 , 3.02–3.69 , 4.32–4.39 , 5.15–5.32 .1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC)  and N,N′-dicyclohexyl-carbodiimide (DCC) , and 4-dimethylaminopyridine (DMAP)  were dissolved in chloroform (10 mL), and then the above mentioned two solutions were mixed together and the mixture was stirred in absence of light at room temperature under nitrogen atmosphere for 24 h. The solution was filtered, and the filtrate was dried under the reduced pressure to remove the solvent. Then the residue was dissolved in chloroform (50 mL) and washed with water (3 × 50 mL), and the organic phase was dried over Na2SO4. The solvent was removed under the reduced pressure, and the crude product was purified by column chromatography  to give PorTaxol as a a purple solid . 1H NMR : δ = −2.79 , 1.18 , 1.30 , 1.72 , 1.73–1.74 , 2.11 , 2.26 , 2.41–2.47 , 2.54–2.55 , 2.57 , 3.89–3.91 , 4.22–4.25 , 4.35–4.38 , 4.50–4.53 , 5.03–5.05 , 5.72–5.74 , 5.88–5.89 , 6.17–6.20 , 6.37 , 6.37–6.43 , 7.19 , 7.36–7.70 , 7.74–7.85 , 8.18–8.22 , 8.32–8.34 , 8.38–8.40 , 8.76–8.77 , 8.86–8.88 ; 13C NMR : δ = 9.7, 15.1, 20.9, 22.7, 25.0, 25.6, 26.9, 29.4, 29.7, 32.0, 34.0, 43.3, 49.2, 58.6, 72.1, 72.2, 75.2, 75.7, 79.3, 81.1, 84.5, 120.5, 126.8, 127.2, 127.9, 128.2, 128.8, 128.8, 129.3, 130.3, 132.1, 133.8, 134.6, 134.9, 142.0, 143.0, 166.0, 167.1, 168.4, 169.9, 171.3, 203.9 ppm; ESI-MS: m/z: 1494.5612 [M+H]+; elemental analysis calcd (%) for C92H79N5O15·8H2O: C 67.43, H 5.84, N 4.27; found: C 67.40, H 5.82, N 4.77.5-(4-carboxyphenyl)-10,15,20- triphenylporphyrin  was dissolved in chloroform (20 mL), and paclitaxel , μmol) in DMSO (500 μL) was added to a solution of HApCD  in deionized water/PBS (25 mL), and then the mixture was ultrasonicated for 5 min. The resulting HATXP solution was dialyzed against excess amount of deionized water for 2 h to remove any unbounded PorTaxol, and the resulting solution was stored at 4 °C. After that the above mentioned solution was extracted by chloroform (3 × 30 mL) until the aqueous phase turned to colorless, then the organic phase was combined and was dried over Na2SO4, and then the chloroform was removed under reduced pressure. The residue was dissolved in 10 mL DMSO, and the concentration of loaded PorTaxol was measured by UV/Vis spectroscopy and the photometric standard curve of PorTaxol at λ = 515 nm.Porphyrin modified paclitaxel  in DMSO (500 μL) was added to a solution of HApCD  in deionized water (25 mL), and then the mixture was ultrasonicated for 5 min. After that, hyaluronidase was added , and the solution was stirred for 2 h at 37 °C. The obtained solution was subjected to the examinations by DLS, TEM and Tyndall effect.Porphyrin modified paclitaxel prodrug  for 24 h. The cells were incubated with Taxol, PorTaxol prodrug, HATXP, HATXP with excess HA, and HApCD . After incubation for 24 and 48 h, the relative cellular viability was measured by the MTT assays. All the data are presented as the mean ± standard deviation.SKOV-3 human ovarian cancer cells were cultured in the McCoy’s 5A medium, and NIH3T3 mouse embryo fibroblasts were cultured in the Dulbecco-modified Eagle’s medium (DMEM), which were both supplemented with 10% fetal calf serum (FCS) in 96-well plates  for 24 h. Then the cells were incubated with HATXP , After incubation for 6 h, the culture medium was removed and the cells were washed with fresh PBS for 3 times, and then subjected to the observation by a fluorescence confocal microscope.SKOV-3 human ovarian cancer cells and NIH3T3 mouse embryo fibroblasts were cultured in the McCoy’s 5A medium, and in the DMEM, respectively, which were both supplemented with 10% fetal calf serum (FCS) in 6-well plates .


On the electrophysiological level, modulations of early  and late ERP components  were observed as a function of problem size and solution correctness. Higher fit children exhibited selective modulations for N170, P3, and N400 amplitude relative to lower fit children, suggesting that fitness influences symbolic encoding, attentional resource allocation and semantic processing during arithmetic tasks. The current study contributes to the fitness-cognition literature by demonstrating that the benefits of cardiorespiratory fitness extend to arithmetic cognition, which has important implications for the educational environment and the context of learning.The current study investigated the influence of cardiorespiratory fitness on arithmetic cognition in forty 9–10 year old children. Measures included a standardized mathematics achievement test to assess conceptual and computational knowledge, self-reported strategy selection, and an experimental arithmetic verification task , which afforded the measurement of event-related brain potentials (ERPs). No differences in math achievement were observed as a function of fitness level, but all children performed better on math concepts relative to math computation. Higher fit children reported using retrieval more often to solve large arithmetic problems, relative to lower fit children. During the arithmetic verification task, higher fit children exhibited superior performance for large problems, as evidenced by greater  Recent research suggests that cardiorespiratory fitness and physical activity (PA) are positively associated with neurocognitive health across the lifespan  for incorrect relative to correct solutions  and event-related potential in particular (ERPs) have proven to be an invaluable tool for evaluating the neural underpinnings of arithmetic cognition  and large (>10) solutions, and afforded the measurement of electrophysiological activity. Furthermore, to assess strategy selection, participants were asked to report how they solved small and large addition problems, which appeared during both the standardized achievement assessment and experimental task. Irrespective of fitness, all children were expected to demonstrate longer RT and decreased ACC for incorrect relative to correct solutions, irrespective of solution size. It was also predicted that all children would demonstrate longer RT and decreased ACC for large relative to small solutions, irrespective of solution correctness; thus replicating prior work  was based on the volume of oxygen consumed during maximum capacity exercise (ml/kg·min−1). Table Forty preadolescent children aged 9–10,  were recruited from the East-Central Illinois region. Participants were bifurcated into higher (>70th percentile) or lower (<30th percentile) fitness groups based on age-specific norms  a plateau in oxygen uptake corresponding to an increase of less than 2 ml/kg·min−1 despite an increase in exercise workload; (2) a peak HR ≥185 beats per minute  activity was recorded from 64 sintered 10 mm Ag-AgCl electrodes , arranged according to the International 10-10 system . Small problems used single-digit operands between 1 and 4 and large problems used single-digit operands between 6 and 9. Ties , and consecutive even operands  were excluded, and the solution was never the product of a × b. For each problem and operand order, both a correct and incorrect solution were created with incorrect solutions being either lesser or greater by 1 than the correct solution. Thus, all incorrect solutions were small split, and parity was controlled.The current arithmetic verification task was modeled on parameters provided by Núñez-Peña and Suárez-Pellicioni . HoweverEach trial consisted of stimuli presented sequentially in the following order: a fixation dot presented for 500 ms, the first operand presented for 1000 ms, a “+” sign presented for 500 ms, the second operand presented for 2000 ms, and then the solution, which was surrounded by a box and remained on the screen until the participant responded or a maximum of 2000 ms elapsed. The inter-stimulus interval was 100 ms and participants were instructed to respond as quickly and accurately as possible. Participants were counterbalanced according to correct response selection, with half of the participants instructed to make a right hand thumb press on a response pad if the solution was correct and the other half instructed to make a left thumb press if the solution was correct. Response assignments were further counterbalanced across fitness groupings. Participants completed two blocks of small problems and two blocks of large problems, which were counterbalanced across participants. Thus, all participants completed 240 trials, 120 for each problem set size, with 60 correct and 60 incorrect solutions presented randomly for each problem set size  - z ] scores were calculated for each problem size.Behavioral data were collected in terms of RT  for correct trials, and ACC (percentage of correct responses) for each task condition. In accord with previous research Geary, , d' . No fitness group differences were realized for math computation, concepts, or composite achievement percentile, .Achievement data are reported in Table m = 2.9 ± 0.5) relative to large problems (m = 2.5 ± 0.7), . However, this effect, was superseded by problem size × fitness interaction, . Post-hoc analysis revealed a significant trend indicating that higher fit children reported relying on retrieval more frequently than lower fit children during large problem solutions, .Analysis of strategy revealed a main effect of problem size, indicating that all participants reported relying more on retrieval than procedural strategies  for small  = 19.90, p < 0.01, η2 = 0.34], and correctness, , indicating that all participants responded more quickly to small (m = 863.58 ± 158.8), relative to large (m = 932.84 ± 166.6) problems, and for correct (m = 838.82 ± 170.9) relative to incorrect (m = 961.8 ± 170.9) problems. Analysis did not reveal any significant effects of fitness, .ACC. Analysis revealed effects of problem size, , and correctness, , which were superseded by a problem size × correctness interaction, . Post-hoc analysis revealed that all participants responded less accurately for large-incorrect problems (m = 74.3 ± 15.9), relative to small-correct (m = 84.7 ± 8.4), small-incorrect (m = 81.7 ± 10.8), and large-correct (m = 81.2 ± 12.7) problems, . No effect of fitness was observed, .d'. Analysis revealed a main effect of problem size, , indicating that all participants were more accurate at detecting correct (and rejecting incorrect) solutions for small (m = 2.7 ± 0.5) relative to large (m = 2.4 ± 0.7) problems. Analysis also revealed a fitness × problem size interaction, . Post-hoc analysis revealed that higher fit (d = 2.7 ± 0.5) relative to lower fit (d = 2.2 ± 0.8) children more accurately detected correct and incorrect solutions only for large size problems, .F = 13.72, p < 0.01, η2 = 0.27], which was superseded by a problem size × region interaction, . Analysis also revealed a problem size × correctness interaction, , and correctness × region interaction, , which were superseded by a problem size × correctness × region interaction, . Post-hoc analysis of the 3-way interaction revealed that P1 amplitude for all participants was greater during small-correct problems over the right ROI (m = 9.2 ± 7.6) relative to the center ROI (m = 6.2 ± 4.6), and during small-incorrect problems over the right ROI (m = 9.7 ± 8.2) relative to the center ROI (m = 6.5 ± 4.8). Further, amplitude during large problems was greater over the right ROI (m = 8.0 ± 4.6) than the center (m = 3.9 ± 3.2) and left (m = 5.7 ± 4.2) ROIs, and amplitude was greater over the left ROI (m = 5.7 ± 4.2) than the center ROI (m = 3.9 ± 3.2), . In addition, amplitude during small-correct problems was greater (m = 6.2 ± 4.5) than for large-correct problems (m = 4.2 ± 3.2) over the center ROI, , and amplitude during small-incorrect problems was greater than large-incorrect problems over the left  and center  ROIs.Amplitude and latency data for the P1 component are presented in Table F = 3.9, p = 0.05, η2 = 0.09], suggesting that lower relative to higher fit children exhibited larger P1 amplitude during incorrect problem solutions. However, post-hoc tests failed to reveal significant effects upon decomposition of the interaction, [t's(38) ≤ 1.87 p's ≥ 0.07].In addition, analysis revealed a fitness x correctness interaction, , which was superseded by a region × correctness interaction, . Post-hoc tests revealed longer latency over the center ROI, for correct and incorrect solutions , relative to the right  and left  ROIs, . Analysis did not reveal any effect of fitness, .Analysis of latency revealed a main effect of region, . Post-hoc analysis indicated that all participants demonstrated greater amplitude over the left (m = −4.9 ± 5.3) relative to the center (m = −2.6 ± 3.8) and right (m = −3.1 ± 4.8) ROIs, [t's(39) ≥ 2.5 p's < 0.02]. Further, a main effect of fitness was revealed, , which was superseded by a fitness × correctness interaction, , η2 = 0.11. Post-hoc tests revealed that higher fit (m = −5.7 ± 4.5) relative to lower fit (m = −1.9 ± 4.3) children demonstrated larger N170 amplitude only during incorrect problem verification, , while no such differences were observed for correct problem verification, . No effects of fitness, problem size, correctness or region were observed for N170 latency, .Amplitude and latency data for the N170 are presented in Table F = 15.30, p = 0.01, η2 = 2.87], which was superseded by a problem size × correctness × region interaction, . Post-hoc tests revealed that all participants demonstrated greater P3 amplitude over the right ROI during small problems (m = 10.7 ± 6.1), relative to the left (m = 7.5 ± 4.5) and center (m = 7.3 ± 4.3), ROIs during large problems, [t's(39) ≥ 3.52], p ≤ 0.01. Further, participants demonstrated greater P3 amplitude over the center ROI during small problems (m = 9.3 ± 5.4) relative to large problems (m = 7.3 ± 4.3), . Further, a fitness × problem size interaction was observed, . Post-hoc tests revealed that higher fit children (m = 8.1 ± 2.8) had smaller P3 amplitude relative to lower fit children (m = 11.4 ± 5.5) during small problems, [t(38) = 2.36], p = 0.02. Lastly, a fitness × correctness interaction indicated that lower fit (m = 10.1 ± 5.3) relative to higher fit (m = 7.3 ± 2.7) children exhibited larger P3 amplitude during incorrect problems, . However, post-hoc tests failed to decompose the interaction, .P3 amplitude and latency data are presented in Table F = 10.50, p < 0.01, η2 = 0.28], indicating that all participants demonstrated longer P3 latency during large (m = 388.2 ± 46.7) relative to small (m = 367.0 ± 43.0) problems. Analysis further revealed an effect of correctness, , indicating that participants had longer P3 latency during incorrect (m = 383.2 ± 48.7) relative to correct (m = 371.9 ± 39.7) problems. An effect of region was also observed, , indicating that P3 latency was longest over the center ROI (m = 393.4 ± 52.7), and shortest over the right ROI (m = 347.6 ± 50.8), with the left ROI (m = 374.8 ± 48.8) falling in-between, . Lastly, a fitness × size × correctness × region interaction was observed, , however, post-hoc tests failed to decompose the interaction, .P3 latency analyses revealed effects of problem size, , and correctness, , which were superseded by a fitness × correctness interaction, . Post-hoc testing revealed that higher fit (m = 0.2 ± 0.9) relative to lower fit (m = 4.4 ± 1.0) children had larger N400 amplitude during incorrect problems, . Analysis based on difference waves (incorrect-correct) confirmed this finding, revealing an effect of fitness, , indicating that higher fit children (m = −2.1 ± 2.0) had greater N400 amplitude than lower fit children (m = −0.3 ± 1.8) during incorrect solutions. No effects of fitness, problem size, ROI, or correctness were observed for N400 latency, .N400 amplitude and latency data are presented in Table a priori predictions, higher fit children reported using retrieval strategies more often for large problems compared to lower fit children; however, all children reported relying more on retrieval strategies for small relative to large problems, suggesting that fitness has a selective relation with specific aspects of arithmetic cognition. Alternatively, no fitness differences were observed for standardized achievement. During the verification task, fitness primarily modulated performance for large problems, but all children demonstrated behavioral modulations as a function of problem size and solution correctness. On the electrophysiological level, both early and late components were modulated by fitness and all participants demonstrated modulations of multiple ERP components as a function of problem size and solution correctness. Thus, these findings extend the current knowledge base of aerobic fitness-related benefits during neurocognitive development and add to a growing body of research detailing the development of arithmetic cognition.The aim of the current study was to extend the literature-base in cardiorespiratory fitness and cognition by assessing strategic, behavioral, and electrophysiological indices of arithmetic cognition in preadolescent children. Consistent with Higher fit children reported greater use of retrieval strategies than their lower fit counterparts during large problem performance, revealing fitness-related differences in strategic deployment as a function of problem size. Beyond fitness, all children reported more frequent retrieval for small relative to large problems. Differences in arithmetic strategy selection are believed to reflect the underlying functional integration of higher-order neurocognitive functions such as memory, visuo-spatial ability, and cognitive control  suggest significant effects may emerge in a larger sample (see Figure Although no specific predictions were made relative to the early ERP components, several notable modulations as a function of fitness and task parameters occurred. First, while the P1 component is typically unevaluated in arithmetic verification paradigms, the current results suggest that fitness, solution correctness, and problem size may modulate P1 amplitude in children (see Figures Post-hoc explanations of these data suggest that fitness may expedite the maturation of arithmetic expertise by facilitating differential numeric encoding of correct and incorrect solutions.Secondly, higher fit children demonstrated greater N170 amplitude than their lower fit peers, and this group difference was found to interact with solution correctness, such that higher fit children demonstrated the greatest amplitude difference during incorrect solution processing (see Figure Both animal (van Praag et al., With respect to later ERP components, lower- relative to higher-fit children exhibited greater P3 amplitude during small problem solutions, with the greatest difference occurring for small-incorrect solutions (see Figures d' scores were positively correlated with N400 amplitude, suggesting that fitness may facilitate the detection of correct solutions and rejection of incorrect solutions via differential activation of semantic memory networks. Indeed, the only other study to evaluate the underlying neurocognitive processes giving rise to greater achievement scores in higher fit children observed a similar finding within the domain of linguistic performance (Scudder et al., In addition to P3 amplitude modulations, higher fit children exhibited significantly greater N400 amplitude to incorrect solutions relative to their lower fit counterparts; a finding further confirmed by difference wave analysis (see Figure While the comprehensive nature of the current study yields valuable information regarding the relation of cardiorespiratory fitness to aspects of arithmetic cognition, it is not without limitations. First, the study design was cross-sectional in nature and it is always possible that some unmeasured variable may have influenced the current results. However, demographic variables such as age, IQ, SES and pubertal timing did not differ between groups and were relatively homogenous between participants. In addition, the relatively small sample size may limit the interpretable power of the current results. Future longitudinal studies with greater sample size will help determine the robustness of the observed effects. Lastly, the current sample was relatively high performing in terms of IQ and academic achievement, potentially limiting the generalizability of the current results.Irrespective of limitations, the findings observed herein add important information to the fitness-cognition literature by revealing that the beneficial effects of fitness extend on the behavioral and neural levels to the domain of arithmetic cognition. The current results provide further incentive for promoting physical activity and fitness in youth, while engendering further inquiry into the relation of fitness and scholastic development. By further detailing strategic, behavioral, and electrophysiological indices of arithmetic cognition during development, the current results also call for a more refined examination of arithmetic development through the evaluation of early ERP components during arithmetic verification as well as the interaction of size and split effects. In summary, the current results add important information to the exercise and arithmetic cognition literatures, illustrating the importance of a physically active lifestyle as well as comprehensive experimental designs when evaluating scholastic development. Lastly, the current results further emphasize the importance of cardiorespiratory fitness during childhood not only for cardiovascular health, but also for neurocognitive and scholastic development.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


B. subtilis MraY (MraYBS). It was found that the simplest analogue of Park’s nucleotide only bearing the first two amino acids, l-alanine-iso-d-glutamic acid, could function as a MraYBS substrate. Also, the acid group attached to the Cα of iso-d-glutamic acid was found to play an important role for substrate activity. Epimerization of the C4-hydroxyl group of muramic acid and modification at the 5-position of the uracil in Park’s nucleotide were both found to dramatically impair their substrate activity. Unexpectedly, structural modifications on the uracil moiety changed the parent molecule from a substrate to an inhibitor, blocking the MraYBS translocation. One unoptimized inhibitor was found to have a Ki value of 4 ± 1 μM against MraYBS, more potent than tunicamycins.Systematic structural modifications of the muramic acid, peptide, and nucleotide moieties of Park’s nucleotide were performed to investigate the substrate specificity of  Peptidoglycan is a polymer consisting of sugars and amino acids that forms the bacterial cell wall. Interrupting the biosynthesis of peptidoglycan can devastate bacterial growth and survival due to the critical role it plays in maintaining cell shape and protecting bacteria from internal osmotic pressure1345Aquifex aeolicus (MraYAA) shows the overall architecture of this interesting enzymeOne major class of MraY inhibitors, known as nucleoside antibiotics, shares a uridine nucleoside as a common moiety with Park’s nucleotide67896 was completely consumed in 1 h when undecaprenyl phosphate (C55P) was applied as a polyprenyl phosphate substrate in our hands  in 2 by treatment with TBAF in THF, followed by coupling with H-d-iso-Glu(OMe)-l-Lys-(TFA)-d-Ala-d-Ala(OMe) and debenzylation gave the corresponding 3 in 67% yield over three steps. Compound 3 was then coupled with activated UMP-morpholine-N,N’-dicyclohexylcarboxamidine salt, followed by global deprotection under basic conditions gave Park’s nucleotide 5 in 35% yield over two steps. A fluorescent probe 6 was prepared from 5 by conjugating a nitronbenzoxadiazole (NBD) fluorophore at the terminal amine site of lysine on the peptide stem in 88% yield. Compounds 7 and 8 were similarly prepared , was only slightly less active than 5 ) and UDP-GlcNAc (10) were not substrates under these assay conditions, showing that this 3-O-lactyl-tripeptide (d-Lac-l-Ala-γ-d-Gln-l-Lys) moiety in Park’s nucleotide is important for the MraYBS catalyzing process.The substrate activity study of activity . Compoun5 were proposed, and the resultant molecules conjugated with a NBD fluorophore on the peptide stem for easy monitoring , and unnatural N-glycinyl 13 had similar substrate activity to 6, indicating that there are no extra interactions, such as additional hydrogen bonds, to increase the activity between the N-substituent moiety on muramic acid of Park’s nucleotide analogues and MraYBS14 (R4 = H) had similar activity to 6, suggesting the methyl group on the lactate moiety to be unessential15 (R5 = H) was slightly less active than 6  was found to be a very poor substrate compared to 6  assay. Initial attempts to perform the MraYBS binding assay in the presence of both substrates  didn’t work properly because a strong non-specific binding signal was observed; presumably, the hydrophobic part of C55 might mainly contribute this non-specific interactionBS. As shown in 5 and 6 exhibited similar binding affinity with KD values of 120 and 127 μM, respectively. This suggests the NBD tag in 6 does not affect the binding affinity with MraYBS, and this observation is consistent with the substrate activity result in 9 is the truncated form of 5 ; and 16 is very similar to 6 – the only difference being removal of the acid group attached to the Cα of iso-d-glutamic acid (R6 = H). However, 9 and 16 showed no proper binding affinity with MraYBS – only a very low binding signal was detected, even at concentrations up to 500 μM. Our results indicate that the acid moiety (R6 = COOH) on iso-d-Glu of Park’s nucleotide plays an important role for both binding affinity and substrate activity. In addition, 11 (R1 = OH/R2 = H) and 17 (R7 = NH2) did not function as substrates, even under extreme reaction conditions, showing the equatorial hydroxyl group at R2 position to be critical and the modifications at R7 position not tolerated.In order to evaluate the effects of peptide moiety of Park’s nucleotide, investigation of the binding affinity of BS is proposed , showing that the mutations caused a loss of binding affinity. The highly conserved threonine (T53) located on loop A is close to the proposed catalytic pocket  and 17 (modified at R7) – neither of which were active substrates – could inhibit the function of MraYBS. To further evaluate the role of positions at R1, R2 and R7, we re-designed and synthesized 18–21 with a truncated peptide  had no inhibition activity, but 19 (R7 = NHAc) showed weak inhibitory activity (Ki = 764 μM) against MraYBS; and 20, bearing a p-tolylacetamide moiety at R7, became a more potent inhibitor (Ki = 11 μM) –strongly indicating that appropriate N-substitution (R7) can enhance inhibitory activity. However, 21, the C4-hydroxyl epimer of 20, was a very poor inhibitor (30% inhibition at 1 mM). This finding implied the C4-hydroxyl epimerization of Park’s nucleotide dramatically impairs both the substrate and inhibitory activities. To improve the inhibitory activity, we reinstalled the tetrapeptide moiety on 20 to give 22, behaving as a competitive inhibitor with the Ki value of 4 μM toward MraYBS, approximately two-fold more potent than tunicamycins (Ki = 9 μM). Our results indicated that (1) analogues 19, 20, and 22 all function as MraYBS inhibitors, even though they all contain a pyrophosphate moiety; and (2) inhibitory potency can be significantly improved through modification of the substituent at the 5-position of the uracil; modification of the oligopeptide moiety can moderately increase the inhibition activity.We were curious whether analogues  peptide . The inheference . As show19–22 were used to evaluate their binding affinity toward MraYBS in the BLI assay  was approximately 1.5 fold stronger than that of 19 . From the modeled complex structure, both inhibitors could exert an additional H-bond interaction with the K226 in the hydrophobic cleft formed by P52, K226, F228, and W297  was approximately 2.3 fold stronger than that of 20, which can be attributed to the contribution of the tetrapeptide moiety.To understand the binding contribution derived from structural modifications, LI assay . The resrefer to  to compe20 and 22 were also investigated and the minimal inhibitory concentrations (MIC) against S. aureus and B. subtilis were determined using standard-broth dilution methods20 and 22 showed no antibacterial activity, even at a high concentration of 200 μM. It may be because the compounds containing the highly charged pyrophosphate moiety were difficult to penetrate the bacterial cytoplasmic membrane22 remains to be explored.The antibacterial activities of BS substrate activity and specificity were evaluated. Our results led to several important findings: (1) the first two amino acids  of the oligopeptide chain are essential for MraYBS recognition; (2) the configuration of the C4-OH on muramic acid is important for MraYBS substrate specificity; and (3) modifications at the 5-position of the uracil dramatically impair the substrate activity  was found to be roughly twice as potent as tunicamycins (Ki = 9 μM) against MraYBS, the first example of Park’s nucleotide-based inhibitors. However, our results not only allow us to infer the minimal structure requirements of Park’s nucleotide as a MraY substrate, but also illuminate a new direction for MraY inhibitor design. And more generally, the concrete nature of these conclusions validate our strategy of systematic substrate structure modifications for the elucidation of enzyme binding site mapping, for membrane proteins without available co-crystal structures. Investigation of the MraY mechanisms, and development of more potent inhibitors with in vivo antibacterial activity are currently ongoing in our laboratory.Unexpectedly, analogues bearing modifications at the 5-position of the uracil were found to be MraY2O, CDCl3 and CD3OD on Bruker AVANCE 600 at ambient temperature. Chemical shifts are given in δ values and coupling constants J are given in Hz. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and dd (double of doublets). High resolution ESI mass spectra were recorded on a Bruker Daltonics spectrometer. Compound 7–22 were prepared as described in All chemicals were obtained from commercial suppliers and used without further purification. All solvents were anhydrous grade unless indicated otherwise. All non-aqueous reactions were performed in oven-dried glassware under a slight positive pressure of argon unless otherwise noted. Reactions were magnetically stirred and monitored by thin-layer chromatography on silica gel. Flash chromatography was performed on silica gel of 40–63 μm particle size. Concentration refers to rotary evaporation. Yields are reported for spectroscopically pure compounds. NMR spectra were recorded on dilute solutions in D1  and 20% Pd(OH)2/C (20 mg) in THF (10 mL) was stirred at RT for 24 h under a hydrogen atmosphere. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated to give a crude intermediate as white solid. The intermediate was used directly without further purification. The solution of intermediate, 1H-tetrazole  and dibenzyl  phosphoramidite  in CH2Cl2/ACN  was stirred at 0 °C for 2 h. The reaction was then cooled to −40 °C and treated with tert-butylhydroperoxide . The reaction was allowed to warm to RT over a period of 1 h. The mixture was diluted with CH2Cl2 (50 mL), and extract with water (20 mL × 2). The organic layers were collected, dried over MgSO4, concentrated, and purified by cc  to afford 2 as a colorless oil . 1H NMR : δ 7.26–7.31 , 6.72 , 6.58 , 5.61 , 4.96–5.05 , 4.31–4.34 , 4.23–4.26 , 4.11–4.14 , 4.02 , 3.84–3.92 , 3.49 , 2.01 , 1.94 , 1.71 , 1.35 , 1.25 , 0.91–0.93 , −0.03 ; 13C NMR : δ 172.4, 172.0, 170.7, 170.6, 169.0, 135.2, 135.0, 128.9 (×2), 128.7 (×4), 129.0 (×2), 128.0 (×2), 96.7, 78.1, 76.6, 70.1, 69.9 (×2), 68.8, 63.8, 61.4, 53.0, 48.1, 22.8, 20.7, 20.6, 18.8, 17.6, 17.1, −1.59 (×3); HRMS calcd for [C37H53N2O14PSi + H]+ 809.3081, found 809.3045.A mixture of 2  and 1.0 M TBAF in THF  in THF (5 mL) was stirred at RT for 2 h. The reaction mixture was concentrated, and the residue was extracted with EtOAc (50 mL), washed with 1.0 N HCl(aq) (50 mL × 2) and water (50 mL × 2). The combined organic layers were dried over MgSO4, and concentrated to afford the monosaccharide intermediate as a colorless oil. The sugar intermediate and PyBOP  in CH2Cl2 (5 mL) were stirred at 0 °C for 5 min. To the above mixture, a solution of H-d-Glu(OMe)-l-Lys(TFA)-d-Ala-d-Ala(OMe) , and DIEA  in THF (5 mL) were added. The reaction mixture was stirred at RT for 30 min. The reaction solvent was removed and the residue was extracted with CH2Cl2 (50 mL), 1.0 N HCl(aq) (50 mL × 2) and water (50 mL × 2). The combined organic layers were dried over MgSO4, filtered, concentrated and purified by cc  to give benzyl protected monophosphate intermediate as colorless oil. A mixture of this intermediate  and 20% Pd(OH)2/C (10 mg) in MeOH (8 mL) was stirred for 1 h under a hydrogen atmosphere. The reaction mixture was filtered through a pad of celite, and the filtrate was concentrated and purified by cc  to give 3 as a colorless oil . 1H NMR : δ 5.45 , 5.06 , 4.36–4.40 , 4.16–4.28 , 4.10 , 3.86 , 3.71 , 3.69 , 2.28–2.30 , 2.20–2.23 , 2.09 , 2.04 , 1.95 , 1.88–2.01 , 1.77–1.80 , 1.70–1.72 , 1.59–1.61 , 1.42 , 1.38 , 1.31 , 1.28–1.45 ; 13C NMR : δ 173.6, 173.5, 173.4, 173.1 (×2), 172.1, 171.9, 171.2, 170.8, 170.3, 157.5, 115.2, 93.9, 78.0, 77.8, 69.2, 68.5, 61.8, 54.4, 54.2, 53.8, 51.5, 51.4, 51.3, 49.4, 48.9, 39.0, 30.8, 30.6, 28.1, 26.7, 22.7, 21.7, 19.6, 19.3, 17.8, 16.6, 16.3, 15.9; HRMS calcd for [C39H61F3N7O21P + Na]+ 1074.3502, found 1074.3518.A mixture of 2  and 20% Pd(OH)2/C (10 mg) in MeOH (8 mL) was stirred for 1 h under a hydrogen atmosphere. The reaction mixture was filtered through a pad of celite, and the filtrate was concentrated and purified by cc  to give 4 as white solid . 1H NMR : δ 5.08 , 4.16–4.32 , 4.10 , 3.86 , 2.11 , 2.06 , 1.95 , 1.40 , 1.31 , 1.01 , 0.06 ; 13C NMR : δ 175.2, 174.1, 173.8, 172.8, 171.8, 95.3, 79.3, 79.0, 70.9, 70.1, 64.8 (×2), 63.4, 55.5, 23.3, 21.2, 21.0, 19.5, 18.3, 17.6, −1.22 (×3). HRMS calcd for [C23H41N2O14PSi−H]− 627.1981, found 627.1996.A mixture of 3 , UMP-morpholine-N,N′-dicyclohexyl-carboxamidine salt , 1H-tetrazole  and 4 Å molecular sieve in anhydrous pyridine was stirred at RT for 24 h under argan. The reaction was concentrated and purified by cc  to give a pyrophosphate intermediate. The intermediate was dissolved in a solution of 1.0 M LiOH(aq)/MeOH  and stirred at RT for the global deprotection. After stirring for 4 h, the reaction was neutralized by 1.0 N HCl(aq), concentrated, and purified by cc (iPrOH/NH4OH(aq) = 2/1, silica gel) to give 5 as white solid . 1H NMR : δ 7.94 , 5.94–5.96 , 5.45 , 4.03‒4.36 , 3.91–3.94 , 3.80–3.87 , 3.77 , 3.62 , 2.98 , 2.28 , 2.12–2.14 , 1.98 , 1.65–1.89 , 1.31–1.44 . HRMS calcd for [C40H65N9O26P2 + H]+ 1150.3589, found 1150.3644.A mixture of 5  and NBD-X-OSu  in a solution of DMF/sat. NaHCO3(aq)  was stirred at RT for 2 h. The solvent was removed and the reaction mixture was purified by cc  to give a fluorescent product. The compound was further purified by a semi-preparative reverse-phase HPLC  with gradient elution of 0.25 M NH4HCO3(aq)/15% MeOH in water (100:0 to 0:100) at a flow rate of 1 mL/min over 45 min, to give compound 6 as a reddish brown solid . The purity of 6 is higher than 95% by analytical anion-exchange HPLC , C55P (200 μM) in 40 μL reaction buffer  were pre-incubated at 37 °C for 15 min. The reactions were initiated by the addition of Park’s nucleotide analogues (10 μM) and incubated at 37 °C for 1 h. The reaction mixtures were added by uridine (0.01 mg/mL) as the internal standard and heated to 100 °C to stop the transferring reaction. The samples were analyzed by RP-C18 HPLC with gradient elution of 0.25 M NH4HCO3(aq)/15% MeOH in water (100:0 to 0:100) at a flow rate of 1 mL/min over 45 min, and the peaks of substrates were monitored at UV 260 nm. For 6 and 11–17, the reaction mixture containing MraYBS (10 μg/mL), C55P (200 μM), 6-hexanoic acid  in 10 μL reaction buffer  were pre-incubated at 37 °C for 15 min. The reactions were initiated by the addition of NBD-Park’s nucleotide analogues (10 μM) and incubated at 37 °C for 1 h. The reaction mixtures were heated to 100 °C to stop the transferring reaction, and samples were analyzed by an anion-exchange column  with a linear gradient elution of NH4OAc (20 mM to 1 M in MeOH) at a flow rate of 1.0 mL/min over 30 min. The fluorescent substrates were monitored with λex 466 nm/λem 535 nm by fluorescence detector. In an extreme reaction condition, which is performed in reaction buffer  containing Park’s nucleotides (10 μM), MraYBS (40 μg/mL) and C55P (1 mM) at 37 °C for 24 h, compounds 11, 17 and 22 were confirmed to not be a substrate.The purified MraY from BS binding was measured in a biolayer interferometry-based binding assay on Octet Red96® instrument . The biotinylation of MraYBS was performed with using EZ-Link® Sulfo-NHS-LC-LC-Biotin Kit . In general, MraYBS (1.7 mg/mL) in 50 μL reaction buffer , was added by the sulfo-NHS-LC-LC-biotin reagent . After incubating for 30 minutes at room temperature, the biotinylated-MraYBS was purified by a ZebaTM Spin Desalting Columns, 7K MWCO , and the buffer was changed to the binding buffer  during the purification. Comparing to the non-biotinylated-MraYBS, the biotinylated-MraYBS remains 62% activity measured by the HPLC-based MraY functional assay in 1 h reaction. The biotinylated MraYBS was then immobilized to Super Streptavidin (SSA) Octet tips  for reaching a fixed signal of 3 nm. Before the assay starting, the MraYBS-labeled SSA sensors were pre-soaked in binding buffer for 30 min. The association of Park’s analogues with MraYBS were measured by incubating MraYBS-labeled SSA sensors in binding buffer containing various concentrations of samples in Octet Red system. The dissociation was monitored by moving the ligand biosenors from the analyte solution to binding buffer. The dissociation constants (KD) were obtained by nonlinear regression analysis using the specific binding model with the GraphPad Prism program .MraYHow to cite this article: Chen, K.-T. et al. Structural Investigation of Park’s Nucleotide on Bacterial Translocase MraY: Discovery of Unexpected MraY Inhibitors. Sci. Rep.6, 31579; doi: 10.1038/srep31579 (2016).


To evaluate the long-term results of chemoradiotherapy (CRT) for stage II-III thoracic esophageal cancer mainly by comparing results of three protocols retrospectively.2 (day 1 and 29) and 5-fluorouracil (5-FU) at 700 mg/m2/24 h (day 1–4 and 29–32) with radiotherapy (RT) of 60 Gy without a break. Patients in Group B received two cycles of CDDP at 40 mg/m2  and 5-FU at 400 mg/m2/24 h  with RT of 60 Gy with a 2-week break. Patients in Group C received two cycles of nedaplatin at 70 mg/m2 (day 1 and 29) and 5-FU at 500 mg/m2/24 h (day 1–4 and 29–32) with RT of 60–70 Gy without a break. Differences in prognostic factors between the groups were analyzed by univariate and multivariate analyses.Between 2000 and 2012, 298 patients with stage II-III thoracic esophageal cancer underwent CRT. Patients in Group A received two cycles of cisplatin (CDDP) at 70 mg/mp < 0.001, p = 0.008 and p < 0.001, respectively). In multivariate analysis, stage, protocol and number of concomitant chemotherapy cycles were significant factors .The 5-year overall survival rates for patients in Group A, Group B and Group C were 52.4, 45.2 and 37.2 %, respectively. The 5-year overall survival rates for patients in Stage II, Stage III (non-T4) and Stage III (T4) were 64.0, 40.1 and 22.5 %, respectively. The 5-year overall survival rates for patients who received 1 cycle and 2 cycles of concomitant chemotherapy were 27.9 and 46.0 %, respectively. In univariate analysis, stage, performance status and number of concomitant chemotherapy cycles were significant prognostic factors (The protocol used in Group A may be an effective protocol of CRT for esophageal cancer. It may be important to complete the scheduled concomitant chemotherapy with the appropriate intensity of CRT. Chemoradiotherapy (CRT) for thoracic esophageal cancer has better local control and overall survival than does radiotherapy (RT) alone and is one of the curative treatments for thoracic esophageal cancer . Some stWe evaluated the long-term results of CRT for stage II-III thoracic esophageal cancer after 2000 mainly by comparing results of three protocols retrospectively. We also evaluated other prognostic factors that influence the results of CRT.Between 2000 and 2012, 298 patients with stage II-III  thoracic esophageal cancer underwent definitive CRT. This study was performed according to the principles of the Declaration of Helsinki (2013). At the time the patients gave their consent for CRT, we did not obtain comprehensive consent including future research study. Because of retrospective study, it is difficult to reacquire agreement from the patients or their family. Therefore, information disclosure is being done to give a chance of participation refusal on home page after Tohoku University School of Medicine Institutional Review Board approved this retrospective study (2014-1-543).Gross tumor volume was defined as the primary tumor and nodal metastasis based on upper gastrointestinal endoscopy, barium swallow, computed tomography (CT) scan and positron emission tomography (PET). If it was difficult to discriminate the primary tumor on RT planning, the clips were placed on the proximal and distal sides of the primary tumor. Initial clinical target volume (CTV) was defined as the region from the supraclavicular to celiac lymph nodes. Initial CTV was made small in consideration of the patient’s general condition. Boost CTV was defined as the primary tumor with a 20–30 mm craniocaudal margin and an approximately 5 mm radial margin and nodal metastasis. Planning target volume was defined as CTV plus a 5–15 mm margin. Basically, the initial CTV received 40 Gy at 2 Gy per day using parallel-opposed anterior-posterior fields. The boost CTV received 20–30 Gy at 2 Gy per day using parallel-oblique fields to avoid the spinal cord. In some cases, dose per fraction was set to 1.8 Gy in consideration of the patient’s general condition and the size of RT fields.2 on day 1 and continuous infusion of 5-fluorouracil [5-FU] at 700 mg/m2 over a 24-h period on day 1–4) with a 4-week intervals and RT dose of 60 Gy. This protocol has been performed since 2009. Patients in Group B received two cycles of chemotherapy (2-h infusion of CDDP at 40 mg/m2 on day 1 and 8 and continuous infusion of 5-FU at 400 mg/m2 over a 24-h period on day 1–5 and 8–12) with a 4-week intervals and RT dose of 60 Gy with a 2-week break after 30 Gy. This protocol has been performed mainly since 2000. Patients in Group C received two cycles of chemotherapy (2-h infusion of nedaplatin [CDGP] at 70 mg/m2 on day 1 and continuous infusion of 5-FU 500 mg/m2 over a 24-h period on day 1–5) with a 4-week interval and RT dose of 60–70 Gy. This protocol has been performed mainly since 2000. The decisions to assign patients to the three protocols was made by experienced clinicians.All patients underwent one of the following three protocols of CRT Fig. . AdjuvanThe primary endpoint of the present study was the 5-year overall survival rate. The secondary endpoints were progression-free survival rate, completion rate of the protocol, pattern of the first relapse and late toxicity.Upper gastrointestinal endoscopy, CT and PET were performed for evaluation of locoregional relapse and distant metastasis every 3–6 months. We described the first treatment at the time of the first relapse.Late toxicities were graded according to the Common Terminology Criteria for Adverse Events version 4.0. An adverse effect more than 90 days after CRT was defined as a late toxicity.p < 0.05. Statistical analysis was performed using JMP® 10 .The characteristics of patients in Group A, Group B and Group C were compared by the 2 × 2 chi-square test for dichotomous variables or the Mann–Whitney test for continuous variables. Overall survival rate and progression-free survival rate were estimated using the Kaplan-Meier method. Differences between patient subgroups for prognostic factors were analyzed using the log-rank test as univariate analysis. Overall survival was measured from the start of RT to the date of death or last follow-up. Progression-free survival was measured from the start of RT to the date of first relapse or death due to any cause. If salvage esophagectomy was performed due to a residual lesion after CRT, we made the date of salvage esophagectomy the date of relapse. Age (66 years or less vs more than 66 years), gender , performance status (PS) (0 vs 1 vs 2), primary site (Upper thoracic esophagus vs Middle thoracic esophagus vs Lower thoracic esophagus), stage (II vs III (non-T4) vs III (T4)), protocol (Group A vs Group B vs Group C), RT dose (60 Gy or less vs more than 60 Gy), number of concomitant chemotherapy cycles (1 cycle vs 2 cycles), and adjuvant chemotherapy (with vs without) were included in the log-rank test. Multivariate analysis was performed using the Cox proportional hazards regression model. All tests were two-sided, and statistical significance was set at the level of p < 0.001, p < 0.001, p = 0.015, p < 0.001, p = 0.019 and p < 0.001, respectively). The median ages of the patients in Group A, Group B and Group C were 67, 66 and 70 years, respectively. The number of patients with PS0/ PS1/ PS2 were 22/24/2, 17/120/10 and 15/57/15, respectively. The numbers of patient with stage II/ stage III (non-T4)/ stage III (T4) in Group A, Group B and Group C were 17/13/18, 47/83/29 and 30/37/24, respectively.The patients’ characteristics are shown in Table The completion rates of RT in Group A, Group B and Group C were 100 % (48/48), 95.0 % (151/159) and 97.5 % (89/91), respectively. Total dose at the cessation of RT was 20–64 Gy , and a total dose of 70 Gy was planned in the prescription for 2 patients. The reasons for cessation of RT were brain infarct in 1 patient, myelosuppression in 2 patients, severe radiation pneumonia (Grade 5) in 2 patients, severe esophageal stenosis in 1 patient, esophagobronchial fistula in 1 patient, infective thrombus in 1 patient, poor general condition in 1 patient, and refusal of RT in 1 patient. The completion rates of 2 cycles of chemotherapy in Group A, Group B and Group C were 79.1 % (38/48), 91.1 % (145/159) and 80.2 % (73/91), respectively. In 19 patients, the dose intensity of chemotherapy in the second cycle was reduced due to myelosuppression and renal dysfunction. Adjuvant chemotherapy after CRT was performed in 67 patients. The number of cycles of adjuvant chemotherapy was 1–8 . The number of patients who received adjuvant chemotherapy in Group A, Group B and Group C who received adjuvant chemotherapy were 14 (29.1 %), 45 (28.3 %) and 8 (8.8 %), respectively.p = 0.082). In univariate analysis, stage, PS and number of concomitant chemotherapy cycles were significant prognostic factors . The 5-year overall survival rates for patients in stage II, stage III (non-T4) and stage III (T4) were 64.0 % , 40.1 %  and 22.5 % , respectively  and adjuvant chemotherapy (p = 0.885). The results of multivariate analysis are shown in Table p < 0.001, p = 0.043 and p < 0.001, respectively). The hazard ratios (HRs) for patients in stage III (non-T4) and stage III (T4) were 2.60  and 4.17 , respectively. The HRs for patients in Group B and Group C were 1.99  and 2.14 , respectively. The HR with 1 cycle of concomitant chemotherapy was 3.17 . We investigated overall survival rates for protocols in each stage just for reference. The 5-year overall survival rates for patients in Stage II in Group A, Group B and Group C were 77.9 % , 68.3 %  and 48.4 % , respectively. The 5-year overall survival rates for patients in Stage III (non-T4) in Group A, Group B and Group C were 53.9 % , 42.0 %  and 39.2 % , respectively. The 5-year overall survival rates for patients in Stage III (T4) in Group A, Group B and Group C were 25.3 % , 19.1 %  and 24.3 % , respectively.The median follow-up period was 23.4 months . A total of 155 patients died during the follow-up period. The 3- and 5-year survival rates in all patients were 51.5 %  and 43.5 % , respectively. Five patients died of second malignancy at 8.5–87.8 months after CRT, and 5 patients died of esophageal hemorrhage at 2.5–11.8 months after CRT. Results of the log-rank tests presented in Table p = 0.130), respectively. The 5-year progression-free survival rates for patients in stage II, stage III (non-T4) and stage III (T4) were 47.3 % , 29.5 %  and 12.8 %  (p < 0.001), respectively. The 5-year progression-free survival rates in patients with 1 cycle and 2 cycles of concomitant chemotherapy were 23.3 %  and 32.7 %  (p = 0.003), respectively. The 5-year progression-free survival rates for PS0, PS1 and PS2 were 43.1 % , 29.5 %  and 15.9 %  (p = 0.032), respectively.The 3-year and 5-year progression-free survival rates in all patients were 35.6 %  and 31.2 % , respectively. The 5-year progression-free survival rates for patients in Group A, Group B and Group C were 46.6 % , 29.0 %  and 28.7 %   and 43.5 % , respectively. The treatment results in the present study were better than those in previous studies, indicating that CRT for stage II-III thoracic esophageal cancer has been improved. Esophagectomy with neoadjuvant chemotherapy in Japan and esophagectomy with neoadjuvant CRT in Western countries have been the main treatments for stage II-III esophageal cancer as previously mentioned –12. HoweLocal relapse rates after CRT were about 30 % in some studies , 7, 16. Although local relapse has been a problem of CRT for esophageal cancer, increasing the complete response rate is an essential requirement to improve the results of CRT. Ishikura et al. reported that 3-year and 5-year overall survival rates were 63 and 51 %, respectively, for complete response patients, whereas 3-year and 5-year overall survival rates were 6 and 2 %, respectively, for non-complete response patients . TherefoTwo cycles of adjuvant chemotherapy after concomitant CRT were performed in many prospective studies , 6–9. AlWith respect to total RT dose, Intergroup (INT) 0123 carried out a clinical trial to compare standard dose RT (50.4 Gy) and high-dose RT 64.8 Gy) combined with CDDP and 5-FU 4.8 Gy co. On the There are some limitations in the present study. First, there were significant differences in some prognostic factors between Group A, Group B and Group C. There was no defined criteria due to the retrospective analysis in the present study. If general conditions were good in less than 80 years patients without renal, cardiac and liver dysfunction, cisplatin-based regimens such as Group A and Group B tended to be performed. Therefore, selection bias may have affected outcomes of CRT in the present study. The patients in Group C were older and the general condition of patients in Group C was poorer than patients in Group A and Group B, as stated above. Second, we did not evaluate overall survival rate in view of smoking and alcohol consumption. Therefore, treatment outcomes and rate of completion of CRT might also have been affected by those factors because the patients, especially those in Group C, might have had some comorbidities caused by those factors. Third, the median follow-up period in Group A was shorter than those in Group B and Group C because the protocol for Group A has been performed since 2009. Therefore, the evaluation of 5-year overall survival rates in the three groups might be inappropriate. However, the 2-year overall survival rate in Group A was better than those in Group B and Group C, and the protocol used in Group A may therefore be an effective CRT protocol be one of for esophageal cancer.CRT for stage II-III thoracic esophageal cancer is effective, and long-term survival can be expected. However, local relapse was observed in many patients. In the future, we may need to select patients having sensitivity to CRT for esophageal cancer more carefully. The protocol used in Group A may be an effective protocol for esophageal cancer. It may be important to complete the scheduled concomitant chemotherapy with the appropriate intensity of CRT. Additional investigation is needed to improve overall survival.