This study examined the effects of a mixture of highly bioavailable omega-3 carboxylic acids (OM3-CA) on nuclear magnetic resonance spectroscopy–assessed lipoprotein particle concentrations and sizes and other cardiovascular risk markers in statin-treated patients with fasting triglycerides (TG) ≥2.3 mmol/L (200 mg/dL) and <5.6 mmol/L (500 mg/dL) and at high cardiovascular risk.After a diet lead-in and statin-stabilization period, 647 patients were randomly assigned to receive capsules of control  4 g/d, OM3-CA 2 g/d (plus OO 2 g/d), or OM3-CA 4 g/d for 6 weeks.p < 0.01) and 4 g/d (p < 0.001), and very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) particle sizes were decreased with both OM3-CA dosages vs. OO . Total VLDL/chylomicron remnant particle concentration was reduced by 8.5 and 16.0 % with OM3-CA 2 and 4 g/d, respectively, vs. a 6.9 % reduction with OO (p < 0.001 for OM3-CA 4 g/d vs. OO). Total HDL particle concentration was also reduced by 1.5 and 3.2 % with OM3-CA 2 and 4 g/d, respectively, vs. a 0.6 % increase with OO (at least p < 0.05 for both comparisons). Changes in total LDL particle concentration were not significantly different for OO vs. OM3-CA at either dosage. Apolipoprotein (Apo) CIII levels decreased by 7.6 and 13.1 % with OM3-CA 2 and 4 g/d, respectively, vs. 3.2 % with OO (p < 0.001 for OM3-CA 4 g/d vs. OO). Lipoprotein-associated phospholipase A2 (Lp-PLA2) mass was reduced by 6.2 and 10.7 % with OM3-CA 2 and 4 g/d, respectively, vs. a 0.1 % increase with OO (p < 0.001 for both vs. OO). There were no significant differences between treatments in high-sensitivity C-reactive protein responses.Compared with OO, low-density lipoprotein (LDL) particle size was increased with OM3-CA 2 g/d (2, in patients with hypertriglyceridemia while taking a statin.OM3-CA were associated with shifts in lipoprotein particle sizes and concentrations, and reductions in Apo CIII and Lp-PLANCT01408303.ClinicalTrials.gov Identifier  The long-chain omega-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been used therapeutically as agents to treat hypertriglyceridemia . A mixtuAPOC3) have been associated with lower TG and reduced coronary and ischemic vascular disease risks [In addition to their lipoprotein lipid–altering effects, omega-3 fatty acids reportedly affect lipoprotein particle subclass sizes and concentrations, including a shift toward an increased proportion of larger, buoyant LDL particles vs. smaller, denser, and potentially more atherogenic, LDL particles –7. Omegase risks –15.2 (Lp-PLA2) and high-sensitivity C-reactive protein (hs-CRP) [The present paper reports analyses from the Epanova combined with a Statin in Patients with hypertRiglycerIdemia to reduce non-HDL cholesTerol (ESPRIT) study, a randomized, controlled, double-blind, parallel-group, phase III clinical trial regarding the effects of OM3-CA at two dosages (2 and 4 g/d), compared with an olive oil (OO) control, on lipoprotein particle and subclass sizes and concentrations, as well as other cardiovascular risk markers, including circulating levels of Apo CIII, and the inflammatory markers lipoprotein-associated phospholipase A(hs-CRP) , in highp < 0.05 or lower). Total cholesterol  and VLDL-C concentrations were also reduced vs. OO with both OM3-CA dosages, and the total-C/HDL-C ratio and Apo AI and Apo B levels were significantly reduced vs. OO with 4 g/d only . LDL-C significantly increased with OM3-CA 2 g/d (4.6 %), compared with OO , but not with OM3-CA 4 g/d (1.3 %). Percent changes from baseline in HDL-C did not differ significantly between OO and either OM3-CA dosage group.The primary results from this study, reported previously , showed As described previously , 647 patp < 0.001) inverse correlation (Spearman’s rho) between changes in TG and LDL particle size in all treatment groups: OO −0.283, OM3-CA 2 g/d −0.244, and OM3-CA 4 g/d −0.242.Baseline, end-of-treatment, and LSM-BT percent change from baseline values for lipoprotein particle sizes and concentrations are presented in Table OM3-CA lowered the concentration of VLDL/chylomicron remnants, but only the OM3-CA 4 g/d response reached statistical significance vs. OO. Large VLDL/chylomicron remnant particles were reduced significantly vs. OO in both OM3-CA groups, medium VLDL particles were reduced significantly vs. OO only in the 4 g/d group, and no significant differences between groups were observed for the small VLDL particle concentration response.Responses in total LDL and intermediate-density lipoprotein (IDL) particle concentrations did not differ significantly between treatments. The concentration of large LDL particles was reduced markedly from baseline in the OO group and reduced to a significantly smaller extent in both of the OM3-CA groups. Small LDL particle concentration was relatively unchanged from baseline in the OO and OM3-CA 2 g/d groups, whereas there was a small decline in the OM3-CA 4 g/d group that reached statistical significance, compared with the OO response.Total HDL particle concentration declined statistically significantly, compared with OO in both OM3-CA dosage groups. Reductions in large and small HDL particle concentrations contributed to these effects, as both were reduced, compared with the responses in the OO group, whereas medium HDL particle concentration increased in both OM3-CA dosage groups vs. OO.2 concentrations changed from 214 (50.2) to 215 (53.3) ng/mL, from 218 (54.7) to 205 (52.5) ng/mL, and from 216 (50.1) to 194 (51.4) ng/mL in the OO, OM3-CA 2 g/d, and OM3-CA 4 g/d groups, respectively. LSM-BT percent changes from baseline in Apo CIII and Lp-PLA2 concentrations are shown in Fig. 2 was significantly reduced from baseline with both the OM3-CA 2- and 4-g/d dosages, compared with the OO response. Mean (standard deviation) hs-CRP concentrations in the OO, OM3-CA 2 g/d, and OM3-CA 4 g/d groups, respectively, changed from 4.2 (5.7) to 4.1 (5.5) mg/L, a LSM-BT reduction of 2.8 %; from 3.9 (6.1) to 3.5 (4.2) mg/L, a 1.2 % increase; and from 4.2 (5.1) to 4.1 (6.1) mg/L, a 4.6 % reduction. LSM-BT percent changes from baseline in hs-CRP did not differ significantly between treatments.Apo CIII decreased from baseline to end-of-treatment mean (standard deviation) concentrations of 15.0 (3.9) to 14.7 (4.1) mg/dL in the OO group, from 15.3 (3.9) to 14.2 (3.9) mg/dL in the OM3-CA 2 g/d group, and from 15.5 (4.0) to 13.4 (3.8) mg/dL in the OM3-CA 4 g/d group. Lp-PLA2, but did not change hs-CRP concentration.In men and women with persistent hypertriglyceridemia while taking a statin, OM3-CA  reduced mean VLDL/chylomicron particle size and concentration, increased mean LDL particle size without altering LDL particle concentration, and reduced mean HDL particle size and concentration. OM3-CA therapy also reduced circulating levels of Apo CIII and Lp-PLAp < 0.05) without a change in LDL particle concentration [In an examination of 4.0 g/d of omega-3 acid ethyl esters (mainly EPA + DHA) in combination with simvastatin 20 mg/d, compared with placebo + simvastatin administered to patients with mixed dyslipidemia, VLDL particle size and concentration decreased, and LDL particle size significantly increased .The present study also adds further evidence that omega-3 therapy lowers circulating concentrations of Apo CIII and Lp-PLAApo CIII , 11, 12  CIII [2 –30 have  CIII [2 , 7. Apo The role that Apo CIII plays in the pathogenesis of hypertriglyceridemia is becoming increasingly evident , 10, 31.2 when added to statin therapy [2 was reduced by 6.2 % and 10.7 % with OM3-CA 2 and 4 g/d, respectively, compared with a change of +0.1 % in the OO group (p < 0.001 for both dosages vs. OO). Of interest is that fenofibrate and niacin increase Lp-PLA2 when added to a statin [2 mass without lowering LDL-C. Lp-PLA2 in circulation is a marker for secretion of PLA2 by macrophages in the arterial wall in response to inflammatory stimuli [2 is uncertain at present, particularly in light of results from two clinical outcomes trials that failed to demonstrate reduced CVD event risk with darapladib, an oral, selective inhibitor of the Lp-PLA2 enzyme [2 in response to omega-3 therapy resulted from reduced secretion of PLA2, reduced association of PLA2 with lipoproteins, or enhanced removal from circulation.Previous studies with 4 g/d of EPA + DHA ethyl esters have shown statistically significant reductions of 5.1 to 11.0 % in Lp-PLA therapy , 7. In ta statin , 39. Ome stimuli . The cli2 enzyme , 42. As hs-CRP is an inflammatory marker, and an elevated level is associated with increased CVD risk , 43. The2, similar to those shown for ethyl ester forms [http://clinicaltrials.gov/show/NCT02104817). Another potential limitation that has been described previously [Although the results herein demonstrate that OM3-CA result in potentially favorable changes in lipoprotein particle concentrations and sizes, Apo CIII, and Lp-PLAer forms –7, they 2, but did not change hs-CRP concentration. The potentially favorable changes in LDL particle size, Apo CIII, and Lp-PLA2 might contribute to reduced CVD risk with OM3-CA therapy; however, this remains to be demonstrated in clinical outcomes trials.In men and women with persistent hypertriglyceridemia while taking statins, OM3-CA  reduced the concentrations and sizes of VLDL/chylomicron remnant and HDL particles and increased LDL particle size without affecting LDL particle concentration. In addition, OM3-CA therapy reduced circulating levels of Apo CIII and Lp-PLAESPRIT was a trial conducted at 96 research sites in the United States. A list of the principal investigators and investigative sites is included in the Subjects included in the study were men and non-pregnant, non-lactating women ≥18 years of age with fasting TG ≥2.3 mmol/L (200 mg/dL) and <5.6 mmol/L (500 mg/dL) (after the statin/diet lead-in), at high risk for a future cardiovascular event, and at or near the NCEP goal for low-density lipoprotein cholesterol (LDL-C)   or on a in situ of the cervix); or clinically important clinical laboratory values at screening.Exclusion criteria included a non-HDL-C level <2.3 mmol/L (90 mg/dL); known lipoprotein lipase impairment or deficiency, Apo CII deficiency, or familial dysbetalipoproteinemia; history of pancreatitis; type 1 diabetes mellitus, use of insulin, or glycated hemoglobin >10 %; poorly controlled hypertension; recent significant nephrotic syndrome or pulmonary, hepatic, biliary, gastrointestinal, or immunologic disease; cancer  on serum obtained from fasting serum samples collected at week 0 (baseline) and week 6 (end of treatment). Apo CIII concentrations were measured using the Randox Apo CIII test, which utilizes an in vitro turbidimetric immunoassay and the Randox Daytona analyzer . Lp-PLA2 mass was determined by a latex particle–enhanced turbidimetric immunoassay on a Roche-P modular analyzer  [Lipoprotein particle subclass concentrations and sizes were analyzed by LipoScience, Inc. , using the Nuclear Magnetic Resonance LipoProfile® method  on serumCA, USA) . hs-CRP 2, and hs-CRP. Efficacy end points for each OM3-CA arm were compared with OO using analysis of covariance, with the baseline value as a covariate and treatment group and statin intensity as factors. Low-intensity statins (defined as those expected to lower LDL-C by <40 % in patients with primary hyperlipidemia) included lovastatin 20–40 mg, pravastatin 10–80 mg, fluvastatin 20–80 mg, simvastatin 10–20 mg, atorvastatin 10–20 mg, and rosuvastatin 10 mg; high-intensity statins included simvastatin 40 mg, atorvastatin 40–80 mg, and rosuvastatin 20–40 mg [p < 0.01. Because rank transformed data cannot be back-transformed into meaningful units, when they were used to generate p-values, models were also run using natural log-transformed values to produce least-squares means–back transformed (LSM-BT) and 95 % confidence intervals (CIs) for response values.SAS  was used for statistical programming and analyses. End points included percent changes from baseline to end of treatment in lipoprotein particles , Apo CIII, Lp-PLA20–40 mg , 48. TheAn exploratory analysis was performed that examined the baseline and changes from baseline in LDL particle size for patients according to their end-of-treatment TG category classified as <1.7 (150), 1.7–2.2 (150–199), or ≥2.3 (200) mmol/L (mg/dL) . Univari


The title compound was produced unexpectedly from (±)-1,2-diphenyl-1,2-propanediol by a sequential non-acid Pinacol rearrangement followed by acetal formation during recrystallization in 1-butanol. The tri-substituted dioxolane ring has a twist conformation and in the crystal, molecules are linked by C—H⋯O hydrogen bonds, forming chains along [001]. 30H28O2, was obtained during recrystallization of (±)-1,2-diphenyl-1,2-propanediol in 1-butanol, from an unexpected non-acid-catalyzed pinacol rearrangement followed by acetal formation of the newly formed aldehyde with the diol. The tri-substituted dioxolane ring has a twist conformation on the C—O bond opposite the methyl-substituted C atom. There is an intramolecular C—H⋯π interaction present involving one of the diphenylethyl rings and an H atom of the phenyl ring in position 4 of the dioxolane ring. In the crystal, molecules are linked by weak C—H⋯O hydrogen bonds, forming chains along [001]. The chains are linked by a second C—H⋯π interaction, forming sheets parallel to the bc plane.The title compound, C The pinacol rearrangement is a well-documented reaction , is illustrated in Fig. 1ca 3σ  of the values found in published X-ray structures °. There is an intramolecular C—H⋯π interaction present involving one of the diphenylethyl rings (C91–C96) and an H atom of the phenyl ring in position 4 of the dioxolane ring  or twist . The envelope flap was most often one of the ring oxygen atoms (309 structures). In the twist structures, the twisted bond was usually either of the O1—C2 type (145 examples), as in the structure described in this paper, or of the O1–C5 type (123 examples). Of the twist structures, there were 39 like the present structure (2), close to an ideal symmetric twist configuration, where the two smallest torsional angles are within 3° of each other.The Cambridge Structural Database  as well as in Willy  al. 1970, that thA B3LYP/6-311+G density functional calculation  are a significant but different distance from the plane through the other three atoms, the conformation can be described as ‘distorted envelope’. When two atoms have equal significant distances in opposite directions from the plane, the ring conformation can be described as ‘twist’, as shown in Fig. 33 groups), has a much larger (O3—C4—C5—O1) torsional angle. The calculated conformation is still that of a twist, but the twist bond in this CH3 model is C4—C5, not O1—C2. The best plane through O1—C2—O3 has C4 + 0.28 Å above the plane and C5 − 0.28 Å below the plane, giving the CH3 model an ideal twist conformation. The DFT-optimized CH3-substituted structure is depicted in Fig. 6To illustrate the conformational properties of the five-membered 1,3-dioxolane ring of the title compound, some B3LYP/6-311+G density functional calculation results -1,2-diphenyl-1,2-propanediol : δ 7.46 , 7.39 , 7.3–7.2 , 7.0-6.6 , 6.44 , 5.83 , 4.95 , 1.97 , 1.83 .The proton NMR spectrum was obtained with a Bruker AVANCE-400 NMR spectrometer using hand-picked crystals having the same morphology as the crystal used for the X-ray study. iso(H) = 1.5Ueq(C) for methyl H atoms, Ueq(C) for methine H atoms, and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015017752/su5202sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015017752/su5202Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015017752/su5202Isup3.cmlSupporting information file. DOI: 1426279CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Heavy metals repress tobacco growth and quality, and engineered nanomaterials have been used for sustainable agriculture. However, the underlying mechanism of nanoparticle-mediated cadmium (Cd) toxicity in tobacco remains elusive.3O4 and ZnO nanoparticles (NPs) on Cd stress in tobacco cultivar ‘Yunyan 87’ (Nicotianatabacum). Cd severely repressed tobacco growth, whereas foliar spraying with Fe3O4 and ZnO NPs promoted plant growth, as indicated by enhancing plant height, root length, shoot and root fresh weight under Cd toxicity. Moreover, Fe3O4 and ZnO NPs increased, including Zn, K and Mn contents, in the roots and/or leaves and facilitated seedling growth under Cd stress. Metabolomics analysis showed that 150 and 76 metabolites were differentially accumulated in roots and leaves under Cd stress, respectively. These metabolites were significantly enriched in the biosynthesis of amino acids, nicotinate and nicotinamide metabolism, arginine and proline metabolism, and flavone and flavonol biosynthesis. Interestingly, Fe3O4 and ZnO NPs restored 50% and 47% in the roots, while they restored 70% and 63% in the leaves to normal levels, thereby facilitating plant growth. Correlation analysis further indicated that these metabolites, including proline, 6-hydroxynicotinic acid, farrerol and quercetin-3-O-sophoroside, were significantly correlated with plant growth.Herein, we investigated the effects of FeThese results collectively indicate that metal nanoparticles can serve as plant growth regulators and provide insights into using them for improving crops in heavy metal-contaminated areas.The online version contains supplementary material available at 10.1186/s12951-022-01509-3. Toxic heavy metals have been attracted public attention for their significant environmental and human health threat , 2. In aNanotechnology has been extensively used in industry, agriculture, and the environment –11. Howe2O3 NPs affected corn seed germination and antioxidant activity. In addition, nano-Fe2O3 acted as nanofertilizers to promote plant growth and enhance photosynthetic efficiency , and lupeol D-maltose, in the roots and/or leaves of Cd-treated tobacco seedlings -11a-hydroxy-9,15-dioxoprost-13-enoic acid in the Cd-treated tobacco roots and/or leaves  metabolites, including lupenone, nicotinic acid adenine dinucleotide, and synephrine, whereas they decreased 4 (2.5%) metabolites, including cantharidin, deethylatrazine, glycyrrhetinic acid, and L-glutamic acid, in the roots and/or leaves  metabolites, including 2 alkaloids , 4 amino acids and their derivatives , and 2 phenols . However, it decreased 4 (1.9%) metabolites, including dihydromyricetin, taraxasterone, and hypotaurine, in the Cd-treated tobacco roots and/or leaves  metabolites, including farrerol, isoalantolactone, and rosmarinine, whereas they decreased 8 (5.5%) metabolites, including 2 diterpenoids , glycyrrhetinic acid, and adenosine 3′-monophosphate, in the roots and/or leaves. In addition, FeSO4 increased, but Fe3O4 NPs decreased the level of officinalisinin I  metabolites, including alpha-d-glucose, aconitine, and cytidine 5'-monophosphate. In contrast, they decreased the levels of 8 (4.4%) metabolites, including 3 alkaloids , dihydromyricetin, and erythritol, in the Cd-treated tobacco roots and/or leaves  metabolites, including l-homoserine, L-threonine, and dexamethasone. In contrast, they decreased 8 (5.1%) metabolites, including 3 flavonoids , 3-carbamyl-1-methylpyridinium 1-methylnicotinamide, and acarbose, in the roots and/or leaves under untreated control conditions  metabolites, including 4 amino acids and their derivatives , 3 nucleotides and their derivatives , and astaxanthin. In contrast, they decreased the levels of 3 (1.5%) metabolites, tacrolimus, 13(S)-HPOT and cedeodarin, in the roots and/or leaves of Cd-treated tobacco seedlings -11a-hydroxy-9,15-dioxoprost-13-enoic acid. Additionally, ZnSO4 increased, but ZnO NPs decreased 2 metabolites, d-proline and l-proline, in the Cd-treated tobacco roots and/or leaves  and sinapyl alcohol in the Cd-treated tobacco roots compared with untreated control , 6-hydroxynicotinic acid (C01020), and L-aspartic acid (C00049) levels, whereas Fe3O4 NPs decreased N1-methylnicotinamide (C02918) levels. ZnO NPs increased nicotinate (C00253) and 6-hydroxynicotinic acid (C01020) levels, while ZnSO4 increased nicotinate (C00253) and L-aspartic acid (C00049) levels in the Cd-treated tobacco roots compared with the untreated control  levels under Cd stress, whereas Fe3O4 NPs or ZnSO4 did not affect its accumulation levels in the Cd-treated leaves compared with the untreated control  in the roots and/or leaves, and these increased macro- and micro-elements were conducive to plant growth, thus improving growth under Cd stress.The mineral element accumulation levels, distribution, and balance play essential roles in maintaining plant growth . We founl-theanine and l-proline, are compatible substances that protect plants against stress [l-theanine, l-histidine, and l-proline levels in the roots and leaves . These primary metabolites promote carbon/nitrogen metabolism, thus enhancing stress tolerance [3O4 or ZnO NPs under Cd stress  and sinapyl alcohol in the roots, while they increased 7 amino acids in the leaves of Cd-treated tobacco seedlings compared with untreated control plants  and ZnO NPs  were purchased from Meilun Biotechnology Co., Ltd. and Macklin Biotechnological Co., Ltd., respectively. Detection was performed using transmission electron microscopy . The hydrodynamic particle size and zeta potentials were determined at 50 mg·L−1 Fe3O4 or ZnO NPs using a dynamic light scattering (LDS) apparatus . Fe3O4 and ZnO NPs stock solutions were prepared according to previous descriptions [3O4 and ZnO NPs concentrations was determined as described previously [Feeviously , 31. The2O in a growth chamber at 26 °C under 16 h light photoperiod for 7 days. Then, 12 consistent-looking seedlings were transplanted to each pot, and the pots were covered with a plastic film that could prevent NPs from entering the solution. The seedlings were fostered in one-fourth (1/4) strength Hoagland solutions for 3 weeks. The solutions replacement was performed every 5 days.The tobacco cultivar ‘Yunyan 87’ was planted at the Yuxi Breeding Base of Yunnan Academy of Tobacco Agricultural Sciences, Yunnan, China. Mature tobacco seeds were air-dried and stored at − 20 °C before use. After being disinfected with 20% (v/v) bleach for 10 min, the seeds were washed with sterile water five times and germinated on Petri dishes with two layers of filter papers soaked in ddH−1 Fe3O4, ZnO NPs, FeSO4 or ZnSO4 with ddH2O every other 3 days for five times, and 1/4 strength Hoagland solutions were added with or without 5 μM CdCl2, which were specified as follows: CK, control; Cd, 5 μM Cd; FeNP, 50 mg·L−1 Fe3O4 NPs; FeNP_Cd, 50 mg·L−1 Fe3O4 NPs + Cd; Fe, 50 mg·L−1 FeSO4; Fe_Cd, 50 mg·L−1 FeSO4 + Cd; ZnNP, 50 mg·L−1 ZnO NPs; ZnNP_Cd, 50 mg·L−1 ZnO NPs + Cd; Zn, 50 mg·L−1 ZnSO4; and Zn_Cd, 50 mg·L−1 ZnSO4 + Cd. Three pots, including 36 seedlings per treatment, were carried out, and about 0.695 mg NPs or ions were exposed to each plant. The solutions were renewed every 5 days, and the seedlings for additional growth for 21 days were harvested.Four-week-old ‘Yunyan 87’ seedlings with consistent growth were used for analysis. Spray bottles were used to perform foliar application of 100 mL freshly prepared 50 mg·LAfter being treated for the indicated time, the plant growth parameters, including plant height, shoot fresh weight (FW), root length and FW, were characterized for each treatment/seedling. First, the plant height was measured from the junction to the shoot apex, and root length was measured to the most extended root tip. Then, the seedlings for each treatment were harvested and weighed to determine the shoot and root FW.2O 5 times, the samples were fixed at 105 °C for one hour and oven-dried till steady weights at 70 °C. Approximately 0.25 mg of fine powders were used for mineral element detection. Subsequently, 10 ml HNO3 and 2 ml HClO4 were successively added and wet-washed overnight, followed by boiling for 2 h at 185 °C. Then, 4 mL of diluted HCl  was added to the residual solution and brought to 25 mL. The mineral elements, including cadmium (Cd), potassium (K), iron (Fe), manganese (Mn), calcium (Ca), copper (Cu), magnesium (Mg), and zinc (Zn), were quantified by ICP-AES.The mineral elements were determined as described previously . BrieflyA total of 60 arrays for the roots and leaves were used for widely targeted metabolomics analyses as described previously , 74 and t-test (IBM SPSS Statistics 20.0) to determine the significant difference between the treatments and the control, and asterisks show significant differences at P < 0.05. For multiple group comparisons, one-way ANOVA followed Tukey’s test was performed, and the different lowercase letters indicated significant differences at P < 0.05.Each experiment was performed using three independent biological repetitions. The results are the means ± standard error (SE). We performed Student’s Additionalfile1:FigureS1. Fe3O4 and ZnO NPs via transmission electron microscopy and dynamic light scattering. Transmission electron microscopy (TEM) imaging of Fe3O4 NPs (a) and ZnO NPs (d), bars=100 nm. Dynamic light scattering (DLS) measurements of Fe3O4 NPs (b) and ZnO NPs (e) particle size distribution by intensity. DLS measurements of zeta potential for Fe3O4 NPs (c) and ZnO NPs (f) dispersed in ddH2O. The different lines represent replicate measurements. The results shown are means ± SE (n=3). FigureS2. Fe and Zn contents were released from different concentrations of Fe3O4 or ZnO NP solutions, respectively. The Fe content in the Fe3O4 NP solutions (a) and Zn content in the ZnO NP solutions (b). FigureS3. OPLS-DA loading plot of metabolites in the roots and leaves of Cd-treated tobacco seedlings. CK, control; Cd, 5 μM Cd. FigureS4. OPLS-DA loading plot of metabolites in the roots among different treatments. CK, control; Cd, 5 μM Cd; FeNP, 50 mg·L-1 Fe3O4 NPs; FeNP_Cd, 50 mg·L-1 Fe3O4 NPs+Cd; Fe, 50 mg·L-1 FeSO4; Fe_Cd, 50 mg·L-1 FeSO4+Cd; ZnNP, 50 mg·L-1 ZnO NPs; ZnNP_Cd, 50 mg·L-1 ZnO NPs+Cd; 50 mg·L-1 ZnSO4; and Zn_Cd, 50 mg·L-1 ZnSO4+Cd. FigureS5. OPLS-DA loading plot of metabolites in the leaves among different treatments. CK, control; Cd, 5 μM Cd; FeNP, 50 mg·L-1 Fe3O4 NPs; FeNP_Cd, 50 mg·L-1 Fe3O4 NPs+Cd; Fe, 50 mg·L-1 FeSO4; Fe_Cd, 50 mg·L-1 FeSO4+Cd; ZnNP, 50 mg·L-1 ZnO NPs; ZnNP_Cd, 50 mg·L-1 ZnO NPs+Cd; 50 mg·L-1 ZnSO4; and Zn_Cd, 50 mg·L-1 ZnSO4+Cd. FigureS6. Venn analysis of differentially accumulated metabolites in the roots and leaves of tobacco seedlings exposed to Cd stress. Cd, 5 μM Cd. Up, upregulated metabolites; Down, downregulated metabolites (Cd/control). FigureS7. Venn analysis of differentially accumulated metabolites in the roots and leaves of tobacco seedlings exposed to Fe3O4 NPs or FeSO4. Four-week-old tobacco seedlings were transferred to 1/4 strength fresh Hoagland solutions, and their foliage was exposed to 50 mg L-1 Fe3O4 NPs (a) or FeSO4 (b) for 21 days. FeNP, 50 mg·L-1 Fe3O4 NPs; Fe, 50 mg·L-1 FeSO4. Up, upregulated metabolites; Down, downregulated metabolites. FigureS8. Venn analysis of differentially accumulated metabolites in the roots and leaves of Cd-treated tobacco seedlings exposed to Fe3O4 NPs or FeSO4. Four-week-old tobacco seedlings were transferred to 1/4 strength fresh Hoagland solutions supplemented with 5 μM CdCl2, and their foliage was exposed to 50 mg L-1 Fe3O4 NPs (a) or FeSO4 (b) for 21 days. Cd, 5 μM Cd; FeNP_Cd, 50 mg·L-1 Fe3O4 NPs+Cd; Fe_Cd, 50 mg·L-1 FeSO4+Cd. Up, upregulated metabolites; Down, downregulated metabolites. FigureS9. Venn analysis of differentially accumulated metabolites in the roots and leaves of tobacco seedlings exposed to ZnO NPs or ZnSO4. Four-week-old seedlings were transferred to 1/4 strength fresh Hoagland solutions, and their foliage was exposed to 50 mg·L-1 ZnO NPs (a) or ZnSO4 (b) for 21 days. ZnNP, 50 mg·L-1 ZnO NPs; Zn, 50 mg·L-1 ZnSO4. Up, upregulated metabolites; Down, downregulated metabolites. FigureS10. Venn analysis of differentially accumulated metabolites in the roots and leaves of Cd-treated tobacco seedlings exposed to ZnO NPs or ZnSO4. Four-week-old tobacco seedlings were transferred to 1/4 strength fresh Hoagland solutions supplemented with 5 μM CdCl2, and foliar exposed to 50 mg·L-1 ZnO NPs (a) or ZnSO4 (b) for 21 days. Cd, 5 μM Cd; ZnNP_Cd, 50 mg·L-1 ZnO NPs+Cd; Zn_Cd, 50 mg·L-1 ZnSO4+Cd. Up, upregulated metabolites; Down, downregulated metabolites. FigureS11. Venn analysis of differentially accumulated metabolites in the roots and/or leaves of tobacco seedlings exposed to Fe3O4 or ZnO NPs. FeNP, 50 mg·L-1 Fe3O4 NPs; ZnNP, 50 mg·L-1 ZnO NPs. Up, upregulated metabolites; Down, downregulated metabolites. FigureS12. Venn analysis of differentially accumulated metabolites in the roots and/or leaves of Cd-treated tobacco seedlings exposed to Fe3O4 or ZnO NPs. Cd, 5μM Cd; FeNP_Cd, 50 mg·L-1 Fe3O4 NPs+Cd; ZnNP_Cd, 50 mg·L-1 ZnO NPs+Cd. Up, upregulated metabolites; Down, downregulated metabolites. FigureS13. Venn analysis of differentially accumulated metabolites in the roots and/or leaves of tobacco seedlings exposed to FeSO4 or ZnSO4. Fe, 50 mg·L-1 FeSO4; Zn, 50 mg·L-1 ZnSO4. Up, upregulated metabolites; Down, downregulated metabolites. FigureS14. Venn analysis of differentially accumulated metabolites in the roots and/or leaves of Cd-treated tobacco seedlings exposed to FeSO4 or ZnSO4. Cd, 5μM Cd; Fe_Cd, 50 mg·L-1 FeSO4+Cd; Zn_Cd, 50 mg·L-1 ZnSO4+Cd. Up, upregulated metabolites; Down, downregulated metabolites. FigureS15. Venn analysis of differentially accumulated metabolites in the roots and/or leaves of tobacco seedlings exposed to Fe3O4 NPs or FeSO4. FeNP, 50 mg·L-1 Fe3O4 NPs; Fe, 50 mg·L-1 FeSO4. Up, upregulated metabolites; Down, downregulated metabolites. FigureS16. Venn analysis of differentially accumulated metabolites in the roots and/or leaves of Cd-treated tobacco seedlings exposed to Fe3O4 NPs or FeSO4. Cd, 5μM Cd; FeNP_Cd, 50 mg·L-1 Fe3O4 NPs+Cd; Fe_Cd, 50 mg·L-1 FeSO4+Cd. Up, upregulated metabolites; Down, downregulated metabolites. FigureS17. Venn analysis of differentially accumulated metabolites in the roots and/or leaves of tobacco seedlings exposed to ZnO NPs or ZnSO4. ZnNP, 50 mg·L-1 ZnO NPs; Zn, 50 mg·L-1 ZnSO4. Up, upregulated metabolites; Down, downregulated metabolites. FigureS18. Venn analysis of differentially accumulated metabolites in the roots and/or leaves of Cd-treated tobacco seedlings exposed to ZnO NPs or ZnSO4. Cd, 5μM Cd; ZnNP_Cd, 50 mg·L-1 ZnO NPs+Cd; Zn_Cd, 50 mg·L-1 ZnSO4+Cd. Up, upregulated metabolites; Down, downregulated metabolites.Additionalfile2:TableS1. The differentially accumulated metabolites in the roots of ‘Yunyan 87’ ( Nicotianatabacum) seedlings.Additionalfile3:TableS2. The differentially accumulated metabolites in the leaves of ‘Yunyan 87’ ( Nicotianatabacum) seedlings.Additionalfile4:TableS3. The Cd-induced differentially accumulated metabolites in the roots of ‘Yunyan 87’ (Nicotianatabacum) seedlings.Additionalfile5:TableS4. The Cd-induced differentially accumulated metabolites in the leaves of ‘Yunyan 87’ (Nicotianatabacum) seedlings.Additionalfile6:TableS5. Correlation analysis of differentially accumulated metabolites in the roots and plant growth parameters .Additionalfile7:TableS6. Correlation analysis of differentially accumulated metabolites in the leaves and plant growth parameters .Additionalfile8:TableS7. Significant correlations between metabolites and growth parameters in both root and leaves.Additionalfile9:TextS1. Supporting information.


Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate various genes involved in cholesterol and fatty acid synthesis. In this study, we describe that naturally occurring isothiocyanate sulforaphane (SFaN) impairs fatty acid synthase promoter activity and reduces SREBP target gene  expression in human hepatoma Huh-7 cells. SFaN reduced SREBP proteins by promoting the degradation of the SREBP precursor. Amino acids 595–784 of SREBP-1a were essential for SFaN-mediated SREBP-1a degradation. We also found that such SREBP-1 degradation occurs independently of the SREBP cleavage-activating protein and the Keap1-Nrf2 pathway. This study identifies SFaN as an SREBP inhibitor and provides evidence that SFaN could have major potential as a pharmaceutical preparation against hepatic steatosis and obesity. The development of metabolic diseases is caused mainly by lipid homeostasis-related disturbances2. Sterol regulatory element-binding proteins (SREBPs), a family of transcription factors including SREBP-1a, SREBP-1c, and SREBP-2, regulate the expression of genes involved in fatty acid and cholesterol biosynthetic pathways3. All three SREBPs are synthesized as inactive precursors located on the endoplasmic reticulum (ER) membrane and are processed to liberate N-terminal halves that function as transcription factors in the nucleus. This proteolytic activation is tightly regulated by the interaction of ER membrane proteins, SREBP cleavage-activating protein (SCAP), and insulin-induced gene (Insig). When intracellular sterol levels are reduced, the SCAP/SREBP complex dissociates from Insig, followed by SCAP binding to the common coated protein II (COPII) proteins Sar1 and Sec23/24, resulting in the incorporation of the SCAP/SREBP complex into COPII-coated vesicles. Consequently, SREBPs enter the Golgi and are processed by site-1 and site-2 proteases4. SREBP-1c expression and proteolytic processing are markedly enhanced in the livers of obese mice6. Furthermore, SREBP-1c overexpression in the liver of transgenic mice causes fatty liver, hypertriglyceridemia, and insulin resistance7. Therefore, abnormal SREBP-1c regulation could be central to obesity-related disease development.Recently, a steady increase has been observed in the number of people suffering from lifestyle-related diseases, such as obesity, dyslipidemia, and type II diabetesS-transferases, and heme oxygenase-18. SFaN-mediated Kelch-like ECH-associated protein 1 (Keap1) cysteine residue modification caused the suppression of Nrf2 degradation, and thereby Nrf2 nuclear translocation and target gene expression stimulation10. SFaN is also reportedly effective in atherosclerosis, diabetes, and obesity treatment13. However, the underlying mechanisms of these SFaN effects are not fully understood.Sulforaphane (SFaN) is an isothiocyanate derived from cruciferous vegetables, such as broccoli, cauliflower, and kale. SFaN is produced by myrosinase-mediated hydrolysis of the glucosinolate glucoraphanin. SFaN reportedly acts through nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor activation to induce phase II detoxification enzymes, such as NAD(P)H-quinone oxidoreductase 1, glutathione FAS) gene promoter region, we have previously reported that several small food ingredient compounds, such as allyl isothiocyanate (AITC), chrysin, and xanthohumol (XN), suppress SREBPs18. In this study, we identified SFaN as a natural food component that reduced SREBP activities and stimulated SREBP precursor form degradation.Using a luciferase reporter gene assay with the fatty acid synthase (FAS promoter (Huh-7/FAS-luc). To promote SREBP activation, Huh-7/FAS-luc cells were sterol-depleted by incubating them with an HMG-CoA reductase inhibitor (fluvastatin). The cells were then treated with approximately 100 naturally occurring food components, and a luciferase assay was performed to evaluate how they affect SREBP activity. As a positive control, we confirmed that 25-hydroxycholesterol (25-HC), a potent SREBP processing inhibitor, markedly reduced the promoter activity in Huh-7/FAS-luc cells , and stearoyl-CoA desaturase (SCD1), and HMG-CoA synthase (HMGCS), HMG-CoA reductase (HMGCR), and squalene synthase (SQS), respectively, decreased with the use of 100 μM SFaN or SFeN  90 destabilizes the SREBP precursor forms preceded by SCAP protein degradation, and this SREBP precursor form destabilization could not be observed in the SCAP-deficient SRD-13A cell line20. This line of evidence led us to consider the possible contribution of SCAP in the SFaN- and SFeN-mediated degradation of the SREBP precursor forms. Figure 19. We confirmed that SFaN and SFeN treatments for 6 h reduced the SCAP and SREBP-1 precursor protein levels in CHO-7 cells -3 × FLAG] and ubiquitin with an N-terminal HA tag (HA-Ub) and cultured in the presence of MG132. Next, SREBP-1a-3 × FLAG was immunoprecipitated with an anti-FLAG antibody and the immunoprecipitates were subjected to immunoblotting using an anti-HA antibody. Although the ladder band signal of ubiquitin-conjugated SREBP-1a was barely detected in the absence of SFaN and SFeN, the SFaN and SFeN treatments intensified it Fig. , suggestTo identify the SREBP-1a region essential for the SFaN- and SFeN-mediated SREBP-1a precursor form degradation and ubiquitination, we generated two expression plasmids encoding ΔN SREBP-1a  and ΔC SREBP-1a . The ΔN SREBP-1a protein was decreased and the ubiquitinated ΔN SREBP-1a was increased by SFaN and SFeN  and ΔC SREBP-1a-(2–784), then treated with SFaN or SFeN. ΔC SREBP-1a-(2–968), and ΔC SREBP-1a-(2–784) were degraded by SFaN and SFeN, whereas ΔC SREBP-1a-(2–594) was not  binding, leading to SREBP-1a ubiquitination. However, the SREBP-1a precursor form was not phosphorylated on Thr426 and Ser430 and was insensitive to Fbw7-dependent degradation26. In this study, we showed that SFaN stimulates ΔN SREBP-1a (amino acids 479–1147) ubiquitination and degradation, which does not contain these amino acid residues , although the specific amino acids have not been identified. Considering that SFaN-mediated degradation of SREBP-1a also occurs in the absence of SCAP , generated non-enzymatically during the brewing process for beer production, also reduced SREBP activity. However, unlike XN, IXN stimulates the ubiquitin–proteasome-dependent SREBP precursor form degradation17. The difference between the SFaN- and IXN-mediated SREBP precursor degradation is their dependence on intracellular cholesterol levels. Although SFaN reduces the SREBP precursor forms regardless of intracellular cholesterol levels  in HFD-fed mice; however, it conversely causes weight gain and the development of insulin resistance in the long-term in HFD-fed mice35. In addition, a decrease in weight gain and improvement in insulin resistance have been observed in Nrf2KO mice38. These results imply that the anti-obesity effect of SFaN cannot be explained by the Keap1-Nrf2 pathway activation alone. As SREBP-1 activity suppression has been reported to reduce body weight and improve insulin resistance in obese model mice39, it is likely that dietary SFaN exerts beneficial effects, at least in part, by the suppression of SREBP-1 activity. Further studies are required to determine whether dietary SFaN suppresses SREBP activity by promoting the SREBP precursor form degradation in the liver of obese mice. SFaN administration also reportedly suppresses adipocyte differentiation, with AMPK activation and decreased peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α expression in the adipose tissue31. More recently, SFaN administration reportedly exerted anti-obesity effects by promoting white adipose tissue browning40. These findings suggest that SFaN administration might exert its effects by regulating multiple pathways in multiple organs.SFaN administration to obesity model animals shows an anti-obesity effect, with a decrease in serum cholesterol and TG levels41, indicating that a longer SFaN treatment period is expected to increase cytotoxicity. Since the degradation of the SREBP precursor forms by SFaN is observed within 3 h of SFaN treatment, it is unlikely that SFaN cytotoxicity is responsible for this effect. We also showed that SFaN treatment with 10 μM SFaN decreased the FAS gene promoter activity and 30 μM SFaN promoted the degradation of the SREBP precursor. Further studies are required to verify whether the similar effects are observed in normal hepatocytes and animal models. Considering that the maximum concentration of SFaN metabolites in serum reaches 7.4 μM when 150 mL of test meal containing 100 g florets of super broccoli is consumed by humans42, it is unlikely that the consumption of broccoli leads to a SFaN serum levels of 30 μM, and dietary supplements containing highly stable SFaN and SFaN derivatives need to be developed. However, it is believed that compounds derived from food that supplemental intake exist at higher concentrations in the intestinal tract than in the serum. The expression of bitter taste-sensing G protein-coupled receptors [type 2 taste receptors (T2Rs)] is regulated by SREBP-2 in enteroendocrine STC-1 cells43. T2Rs are known to sense dietary toxins44 and promote cholecystokinin secretion45, thereby suppressing food intake. Therefore, it has been hypothesized that T2Rs play a role in limiting toxin absorption. We demonstrated that SFaN promotes SREBP-2 precursor degradation and thereby reduces their activity. Thus, SFaN consumption might downregulate the expression of bitter taste receptors in the intestinal tract and affect the sensing of dietary toxins.Here, we analyzed the effects of SFaN using human hepatoma Huh-7 cells. We demonstrated that 30 and 100 μM SFaN treatment for 3 h did not affect the viability of Huh-7 cells as well as LDH release from Huh-7 cells, whereas 20–60 μM SFaN treatment for 24 h reportedly decreased the viability of Huh-7 cellsIn summary, the present data demonstrate that SFaN reduces SREBP activity by promoting the degradation of SREBP precursors. Furthermore, we showed that C-terminal region is crucial for SFaN- and SFeN-mediated SREBP-1a precursor form degradation. Additional studies are required to elucidate the direct SFaN targets of these effects.4Cl were purchased from Sigma . Dulbecco’s modified Eagle’s medium (DMEM) was from Wako . Blasticidin S was from Invitrogen . SFaN and SFeN were from LKT Laboratories .Cholesterol, 25-HC, CHX, fluvastatin, lipoprotein-deficient serum, MG132, and NH46. Peroxidase-conjugated affinity-purified donkey anti-mouse IgGs and peroxidase-conjugated affinity-purified donkey anti-rabbit IgGs were purchased from Jackson ImmunoResearch Laboratories .Monoclonal anti-SREBP-1 (2A4), anti-SCAP (9D5), and anti-HSP90 (F-8) antibodies were purchased from Santa Cruz . Monoclonal anti-FLAG (M2) and anti-β-actin (AC-15) antibodies were purchased from Sigma . Monoclonal anti-HA (16B12) antibody was purchased from BioLegend . The polyclonal anti-SREBP-2 (RS004) antibody has been previously describedMedium A contained DMEM supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Medium B was medium A with 10% (v/v) fetal bovine serum (FBS). Medium C was medium A with 10% FBS, 50 μM sodium mevalonate, and 12.5 μM fluvastatin. Furthermore, medium D was medium A with 5% (v/v) lipoprotein-deficient serum, 50 μM sodium mevalonate, and 12.5 μM fluvastatin. Medium E was medium A with 10% FBS, 10 μg/ml cholesterol, and 1 μg/ml 25-HC. Medium F contained DMEM/Ham’s F12 supplemented with 5 μg/ml cholesterol, 1 mM sodium mevalonate, 20 μM sodium oleate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5% FBS.19. Huh-7 cells and HEK293 cells were maintained in medium B. Huh-7/FAS-luc cells (a stable Huh-7 cell line expressing a luciferase reporter driven by an SRE-containing FAS promoter)47 cells were maintained in medium B containing 2 μg/ml blasticidin S. CHO-7 cells and SRD-13A cells were maintained in medium F. SRD-13A cells were originated from the Brown and Goldstein Laboratory19. The cells were incubated at 37 °C under a 5% CO2 atmosphere.Huh–7 cells and HEK293 cells were obtained from ATCC. CHO-7 cells and SRD-13A cells, an SCAP-deficient line derived from CHO-7 cells, were kindly provided by Debose-Boyd RA 5 cells/well, cultured with medium B for 24 h, and the cells were then switched to medium C for 16 h. After incubation for another 24 h in the absence or presence of 10 μM SFaN or SFeN, 30 μM SFaN or SFeN, or 100 µM SFaN or SFeN, the luciferase activity and protein contents of the cell extracts were measured as described previously48. The normalized luciferase values were determined by dividing the luciferase activity by the protein content in the cell extracts quantified using the BCA protein assay (Pierce).Huh-7/FAS-luc cells were plated in 12-well plates at a density of 1.0 × 1049 using Cell Counting Kit-8 (Dojindo) and Cytotoxicity LDH Assay Kit-WST (Dojindo), respectively. Huh-7 or Huh-7/FAS-luc cells were plated in 96-well plates at a density of 2 × 104 cells/well and cultured with medium B for 24 h. After a 3-h incubation in the absence or presence of 30 or 100 μM SFaN, assays were performed following to the manufacturer’s instructions.Cell viability and the plasma membrane damage assays were performed as described previously18. Total RNA was extracted from Huh-7 cells using ISOGEN  according to the manufacturer’s instructions. RNA was reverse transcribed using a high capacity cDNA reverse transcription kit . Real-time quantitative PCR (Taqman probe and SYBR green) analysis was performed on StepOnePlus Real-Time PCR Systems. Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The TaqMan ID numbers for genes analyzed are as follows: human (h) FAS, Hs00188012_m1; hSCD1, Hs00748952_s1; hGAPDH, 4352934; The sequences of the primer sets used were as follows: hACC1, 5′-TGGGCCTCAAGAGGATTTGT-3′ and 5′-TCCACTGTTGGCTGATACATAGATG-3′; hHMGCS, 5′-GACTTGTGCATTCAAACATAGCAA-3′ and 5′-GCTGTAGCAGGGAGTCTTGGTACT-3′; hHMGCR, 5′-TACCATGTCAGGGGTACGTC-3′ and 5′-CAAGCCTAGAGACATAATCATC-3′; hHSP27, 5′-CACGAGGAGCGGCAGGACGAG-3′ and 5′-CAGTGGCGGCAGCAGGGGTGG-3′; hNrf2, 5′- TACTCCCAGGTTGCCCACA-3′ and 5′-CATCTACAAACGGGAATGTCTGC-3′; hSQS, 5′-ATGACCATCAGTGTGGAAAAGAAG-3′ and 5′-CCGCCAGTCTGGTTGGTAA-3′.Real-time quantitative PCR was performed as described previously18. Cells and mouse liver were lysed in RIPA buffer  supplemented with a protease inhibitor cocktail. The lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane , and probed with the antibodies indicated in the figure legends. The immunoreactive proteins were visualized using ECL  or Immobilon  immunoblotting detection reagents. The signals on the membrane were detected by ImageQuant LAS 4000 mini . Full western blot images are presented in the Immunoblotting was performed as described previously50.Expression plasmids containing C-terminal FLAG-tagged SREBP-1a-(2–1147), SREBP-1a-(479–1147), SREBP-1a-(2–594), SREBP-1a-(2–784), and SREBP-1a-(2–968) were constructed by ligating NotI-XbaI PCR fragments encoding the corresponding amino acid regions of human SREBP-1a into the pCMV14-3 × FLAG vector (Sigma) in-frame with the C-terminal FLAG tag. The PCR primer sequence sets used were as follows: SREBP-1a-(2–1147), 5′-ATATGCGGCCGCGATGGACGAGCCACCCTTCAGCGAG-3′ and 5′- ATATCTAGAGCTGGAAGTGACAGTGGTCC-3′; SREBP-1a-(479–1147), 5′-ATATGCGGCCGCGATGAGCCGGGGCATGCTGGACCG-3′ and 5′- ATATCTAGAGCTGGAAGTGACAGTGGTCC-3′; SREBP-1a-(2–594), 5′-ATATGCGGCCGCGATGGACGAGCCACCCTTCAGCGAG-3′ and 5′- ATATCTAGAGGCCAGGTCCAGGTCAGCCT-3′; SREBP-1a-(2–784), 5′-ATATGCGGCCGCGATGGACGAGCCACCCTTCAGCGAG-3′ and 5′-ATATCTAGACCATGGGGTACTGAGCACGGA-3′; and SREBP-1a-(2–968), 5′-ATATGCGGCCGCGATGGACGAGCCACCCTTCAGCGAG-3′ and 5′-ATATCTAGAGGCCTTGTCAATGGAGCTGCT-3′. An expression plasmid containing the C-terminal FLAG-tagged full-length Keap1 (1–625) was constructed by inserting PCR fragments encoding the corresponding amino acid regions of human Keap1 into the BamHI site of the pCMV14-3 × FLAG vector in-frame with the C-terminal FLAG tag using In-fusion HD Cloning kit (Takara) following to the manufacturer’s instructions. The PCR primer sequence sets used were as follows: Keap1 (1–625), 5′-CGACTCTAGAGGATCCATGCAGCCAGATCCCAGGCC-3′ and 5′-AGTCAGCCCGGGATCCACAGGTACAGTTCTGCTGGT -3′. The expression plasmid containing HA-ubiquitin (HA-Ub) was a gift from Edward Yeh (Addgene plasmid # 18712)51. siRNAs for human HSP27 (#6568) and control siRNAs (#6526) were obtained from Cell Signaling Technology. siRNA for human Nrf2 (s9492) was obtained from Ambion; control siRNA (pGL2 luciferase) was obtained from Bonac. Huh-7 cells were transfected with siRNA (24 pmol per six-well plate) using lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions.siRNA experiment was performed as described previously18. Huh-7 cells were plated in 100-mm dishes at a density of 2 × 106 cells/dish and cultured with medium B for 24 h. The cells were then transfected with 15 μg of plasmids including 12 μg of pCMV-SREBP-1a-3 × FLAG and 3 μg of HA-Ub. After incubation for 24 h, the cells were trypsinized and seeded in 100 mm dishes and cultured further under the same conditions. After a 24-h incubation, the cells were treated with 10 μM MG132 for 30 min. After incubation for another 3 h in the absence or presence of 100 μM SFaN or SFeN, the cells were harvested and lysed with Nonidet P-40 lysis buffer  supplemented with a protease inhibitor cocktail. The cell lysate was passed through a 25-gauge needle 20 times, rotated at 4 °C for 1.5 h, and centrifuged at 20,000×g for 30 min. The supernatant was rotated overnight with anti-FLAG M2 Affinity gel (Sigma) at 4 °C, and the pelleted gel was washed 3 times with Tris-buffered saline (TBS). The bound proteins were eluted with 3 × FLAG peptide and were subjected to immunoblotting.Immunoprecipitation was performed as described previouslyN-(4-mercaptobutyl)phthalimide (2) was prepared according to the reported procedure by Ren’s group52.2  in THF (13 mL) were added NaH  and 4-bromobut-1-yne  at room temperature. After stirring for 2 h at room temperature, the reaction mixture was poured into water at 0 °C and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was subjected to silica gel column chromatography (Hex/EtOAc = 10:1) to give N-(4-(but-3-ynylthio)butyl)phthalimide .Under Ar atmosphere, to a solution of thiol 3: 1H NMR : δ (ppm) = 1.64 , 1.79 , 2.02 , 2.47 , 2.61 , 2.68 , 3.70 , 7.71 , 7.84 .Compound 3  in MeOH (5 mL) was added hydrazine monohydrate  and the mixture was refluxed for 3 h. Then conc. H2SO4 (200 µL) was added to the reaction mixture, which was refluxed for further 1 h. After cooling down to room temperature, water (6.5 mL) was added to the mixture, and the resulting precipitate was removed by filtration. Solvent was evaporated in vacuo to give crude 4-(but-3-yn-1-ylthio)butan-1-amine (80 mg), which was used in the next reaction without further purification. This crude amine (80 mg) was dissolved in CH2Cl2 (1.7 mL) and treated with di-2-pyridyl thionocarbonate , followed by 5% aqueous NaOH solution (1.2 mL). The mixture was stirred for 4 h at room temperature, then diluted with H2O, and extracted with CHCl3. The combined organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was subjected to silica gel column chromatography (Hex/EtOAc = 10:1–3:1) to give but-3-yn-1-yl(4-isothiocyanatobutyl)sulfane  as a pale yellow oil.Under Ar atmosphere, to a solution of phthalimide 4: 1H NMR : δ (ppm) = 1.70–1.86 , 2.05 , 2.49 , 2.62 , 2.70 , 3.56 .Compound 4  in CH2Cl2 (2.7 mL) was slowly added m-chloroperoxybenzoic acid  at – 78 °C. The solution was After stirring for 1 h at − 78 °C, saturated sodium bicarbonate solution and 10% aqueous sodium thiosulfate solution were added to the reaction mixture and the mixture was extracted with CH2Cl2. The combined organic layer was washed with saturated sodium bicarbonate solution and brine successively, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was subjected to silica gel column chromatography (Hex/EtOAc = 3:1–1:2) to give 4-((4-isothiocyanatobutyl)sulfinyl)but-1-yne  as a colorless oil.Under Ar atmosphere, to a solution of sulfide 1: IR (film): νmax (cm-1) = 2179, 2100, 1024. 1H NMR : δ (ppm) = 1.84–2.01 , 2.09 , 2.70–2.86 , 2.89 , 3.61 . HRMS (ESI-TOF): m/z calcd. for C9H13NNaOS2 [M + Na]+ 238.0331, found 238.0340.Compound 53, with minor modifications. Immobilization was performed with FG beads  according to the manufacturer’s instructions, with modifications. Azide beads (1 mg) were incubated with 62.5 μM Tris[methyl]amine, 1.25 mM CuSO4, 1.25 mM ( +)-Sodium l-ascorbate, and 125 μM SFaN-Alkine-derivative in tert-butyl alcohol/DMSO (4:1) for 16 h at room temperature. The beads were collected by centrifugation, washed three times with tert-butyl alcohol/DMSO/H2O (4:1:5), then washed three times with MeOH/H2O (1:1) and stored in MeOH/H2O (1:1) at 4 °C.SFaN beads preparation was performed according to a previously published procedure5 cells/dish and cultured with medium B for 24 h. The cells were then transfected with 1 μg of pCMV-SREBP-1a-(2–1147) -3 × FLAG or pCMV-Keap1-3 × FLAG plasmid. After incubation for 24 h, cells were lysed in NP-40 buffer  supplemented with a protease inhibitor cocktail. The lysed cells were centrifuged at 20,000×g. The supernatant was collected and incubated with the SFaN beads under conditions with or without 100 μM SFaN for 1.5 h at 4 °C. After collecting the beads magnetically and washing them four times with wash buffer , SDS sample buffer was added to the beads, and then the suspensions were heated at 96 °C for 5 min. The elution samples were subjected to immunoblotting.HEK293 cells were plated in 6 well plate at a density of 5 × 10t test for two groups. One-way ANOVA followed by the Bonferroni procedure was used to compare more than two groups. Differences were considered significant at P < 0.05.All data are represented as mean ± S.E. Statistical analysis was performed using the Ekuseru-Toukei Ver.2.0 . Comparisons between treatments were made by a Student’s Supplementary Figures.


Their structures were fully determined by extensive spectroscopic analyses, including ECD, and X-ray crystallography in case of compound 5, whose absolute configuration was determined for the first time. The isolates 1, 6–14 were evaluated for their anti-inflammatory and anti-fungal activity against three skin fungi, e.g., Epidermophyton floccosum, Trichophyton rubrum, and Microsporum gypseum. Most of the isolates showed weak anti-fungal and anti-inflammatory activity. Only compound 9 exhibited obvious anti-fungal activity against E. floccosum (MIC50 = 2.467 ± 0.03 μM) and M. gypseum (MIC50 = 4.673 ± 0.077 μM), while the MIC50 values of the positive control terbinafine were 1.287 ± 0.012 and 0.077 ± 0.00258 μM, respectively. The results indicated that simple emodin type anthraquinone is more potential against skin fungi than its oxyglucosyl, C-glucosyl and glycosylated seco analogues.A new seco-anthraquinone, crispuside A (The online version contains supplementary material available at 10.1007/s13659-022-00350-3. Rumex with more than 200 species distributing widely in the world is the second largest genus in the family Polygonaceae, in which some species displayed nutritional and medicinal properties. For example, Rumex patientia L. was reported having abundant amino acid and cellulose  and a 3-H singlet signal at δH 2.38  due to a methyl group were observed, together with one anomeric proton at δH 4.85  and a set of proton signals in the range between δH 2.3–3.8, disclosing the existence of a sugar moiety. The J1″−2″ coupling constant of anomeric proton (7.8 Hz) revealed the glucosyl anomeric center to be β configuration. The 1H and 13C NMR data of 1 were closely related to those of 6 , two methylenes , one aromatic methine , two oxymethines , one carbonyl group , and a set of quaternary aromatic carbons . These spectroscopic features were similar to those of -9-hydroxy-O-methylasparvenone  −, and 13C NMR and DEPT spectroscopic data. The 13C NMR spectrum of 5 showed one carbonyl (δC 208.6), six aromatic carbon signals  with three methines and one oxygenated quarternary carbon, and six sp3 carbon signals  assignable to two methyls, two oxymethines and two oxy quarternary carbons. The 1H NMR spectrum showed three characteristic proton resonances at δH 6.72 , 7.17 , 7.10  suggesting the existence of a 5,9,10-trisubstituted benzene ring. In addition, two methyls  and two oxymethines  signals were observed. The above data indicated that 5 was a naphthoquinol derivative, whose epoxide ring was inferred by chemical shifts  and seven degrees of unsaturation. The detailed analyses of the 2D NMR  spectra   at a concentration of 100 μM (Table S1), as previously described −; HRESIMS m/z 449.1094 [M−H]−, .Yellowish amorphous powder; UV (MeOH) c 0.07, MeOH); UV (MeOH) λmax (log ε) 222 (3.1) 270 (2.8), 330 (2.3) nm. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table m/z 235 [M−H]−; HRESIMS m/z 235.0979 [M−H]−, .White amorphous powder, entclass1pt{minimac 0.16, MeOH); UV (MeOH) λmax (log ε) 222 (3.1), 270 (2.8), 330 (2.3) nm. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table m/z 235 [M−H]−; HRESIMS m/z 235.0979 [M−H]−, .White amorphous powder; entclass1pt{minimac 0.76, MeOH); UV (MeOH) λmax (log ε) 240 (3.1), 330 (2.6) nm. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table m/z 233 [M−H]−; HRESIMS m/z 233.0814 [M−H]−, .Yellow amorphous powder; entclass1pt{minima1H (500 MHz) and 13C (125 MHz) NMR (in methanol-d4) data, see Table m/z 249 [M−H]−.White amorphous powder; and 13C 15 MHz NMR5: C13H14O5, M = 250.24, a = 7.7634 (3) Å, b = 8.2971 (3) Å, c = 9.2218(3) Å, α = 90°, β = 107.7570 (10)°, γ = 90°, V = 565.71 (4) Å3, T = 100 (2) K, space group P1211, Z = 2, μ(Cu Kα) = 0.954 mm−1, 10,180 reflections measured, 2156 independent reflections (Rint = 0.0500). The final R1 values were 0.0319 [I > 2σ(I)]. The final wR(F2) values were 0.0821 [I > 2σ(I)]. The final R1 values were 0.0333 . The final wR(F2) values were 0.0832 . The goodness of fit on F2 was 1.064. Flack parameter = 0.17(10). Crystallographic data for the structure of 5 have been deposited in the Cambridge Crystallographic Data Centre . Copies of the data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk.Colorless crystal of 1 and 6–14 were evaluated for their anti-fungal against three skin fungi  and anti-inflammatory activity. For anti-fungal activity, Terbinafine hydrochloride was used as positive control. Fungal broth (5 × 105 CFU mL−1) and test samples (100 μM) were incubated in 96-well plates at 25 °C for 5 days, A microplate reader was recorded by the absorbance at 625 nm. The experiment also set up the culture medium blank control, fungi control and terbinafine hydrochloride positive drug control.The isolates 1 and 6–14 was screened as previously reported method [1 and 6–14  was added, and no drug group and L-NMMA positive drug group were taken as controls. After the cells were cultured overnight, the medium was used to detect NO production and the absorbance was measured at 570 nm. MTS was added to the remaining medium to detect the cell survival rate and exclude the toxic effects of the compounds. The formula to calculate the inhibition rate is as follows: NO production inhibition rate (%) = (non-drug treatment group OD570 nm − sample group OD570 nm)/non-drug treatment group OD570 nm × 100%.The anti-inflammatory activity of d method . The mou1–4) and ten known (5–14) quinone derivatives were isolated and identified from the roots of Rumex crispus L. Compound 1 is a seco-anthraquinone glucoside, while 2–4 belong to naphthalenones containing 3,4-dihydronaphthalen-1(2H)-one moiety. The absolute configuration of 5 was determined for the first time by X-ray single crystal diffraction. The anti-fungal and anti-inflammatory activity of anthraquinones  was tested, of which compound 9 showed obvious anti-fungal activity. The results indicated that simple emodin type anthraquinone is more potential against skin fungi than its oxyglucosyl, C-glucosyl and glycosylated seco analogues.Four new (Additional file 1. Supporting information.


Z′ = 2. Water bridges the gap between phosphocholinehead groups caused by the interdigitated bilayer structure. The twomethanol solvates are also mutually isostructural with the head groupsadopting a more extended conformation. Again, the solvent bridgesthe gap between head groups in the bilayer. No anhydrous form of miltefosineor its analogues were isolated, with dehydration resulting in significantlyreduced crystallinity. This arises as a result of the integral rolethat hydrogen-bond donors (in the form of water or solvent molecules)play in the stability of the zwitterionic structures.Miltefosine is arepurposed anticancer drug and currently the onlyorally administered drug approved to treat the neglected tropicaldisease leishmaniasis. Miltefosine is hygroscopic and must be storedat subzero temperatures. In this work, we report the X-ray structuresof miltefosine monohydrate and methanol solvate, along with 12- and14-carbon chain analogue hydrates and a solvate. The three hydratesare all isostructural and are conformational isomorphs with  Hydrogen-bond donors in the form of wateror solvent moleculesplay a key role in the stability of the antileishmaniasis drug miltefosine. No single-crystalX-ray diffraction (SC-XRD) structures of PC16 or closely related analogueswith different alkyl chain lengths are currently reported in the CambridgeStructural Database (CSD);20 however, arelated, chiral glycerol-derived phosphocholine structure with an18-carbon alkyl chain (3-octadecyl-2-methyl-d-glycero-1-phosphocholine) is known and also exists as a monohydrate (refcode:DONZAH), suggesting that there may be a consistent structural reasonfor hydrate formation in this class of compound.21 In DONZAH, the molecules pack in a bilayer with a herringbonepattern with interdigitating head groups and hydrocarbon chains, alongsidehydrogen bonds from the water molecule to the phosphate oxygen atomat an O···P distance of 2.80 Å and a Z′ of 1.21Miltefosineor P1̅) with two crystallographicallyindependent molecules of miltefosine and two water molecules. Thestructure is thus a monohydrate with Z′ =2  and 2.812(6) Å from the hydroxy group of water to thephosphate oxygen atom of the PC16 headgroup.To obtain the crystalsof PC16, a sample was dissolved in 0.5 mL of chloroform and toluene(1:1) and was sonicated at 70 °C for 1 min and allowed to coolto room temperature, which yielded colorless birefringent block crystalsafter 3 weeks. The SC-XRD determination revealed a centrosymmetrictriclinic structure , as shown in A separate crystallization experiment of PC16 resulted in the formationof colorless needle crystals from the slow cooling of 2-butanol after3 weeks. The SC-XRD analysis revealed a (disordered) methanol solvateof PC16. Methanol appears to arise from inadvertent vapor diffusionfrom adjacent samples. This PC16 solvate is also a Both of these miltefosine structures implythat the presence ofa solvent molecule is necessary to fill gaps between the polar groupsleft by the bilayer structure. In order to probe the generality ofhydrate and solvate formation in this class of compound, we examinedthe structures of closely related analogues of PC16. We also exploredthe possibility of producing an anhydrous form of PC16 miltefosineitself.n-tetradecylphosphocholine(PC14) and n-dodecylphosphocholine (PC12). PC14 alsocrystallizes as both a hydrate and a methanol solvate (Z′ = 2) with a shorter c axis reflectingthe shorter alkyl chain, and again water plays an integral role inholding the PC14 molecules together in a hydrogen-bonded chain thatspans the width of the interdigitated bilayer. Hydrogen-bonded OH···Odistances are 2.760(4) and 2.808(3) Å. The PC14 methanol solvateis also isostructural to its PC16 analogue  with the same more extended conformations,although in this case most of the methanol is ordered, while one ofthe PC14 molecules in the asymmetric unit is twofold disordered. Twoordered methanol molecules are situated in discrete pockets hydrogenbonding to two of the phosphate oxygen atoms of just one of the twoPC14 molecules which has a more extended conformation, with OH···Odistances of 2.685(4) and 2.707(4) Å. The other PC14 moleculehas a more compact conformation and accepts a hydrogen bond from afurther disordered methanol molecule. In addition, there is a smalllattice void that is occupied by an additional partially occupiedmethanol molecule.In addition to PC16, the SC-XRD structures of two shorter-chainanalogues were also determined, namely,  solvate 3a, from The short-chain analogue PC12 was slowly cooled in acetonitrileand yielded plate crystals after 5 days. The SC-XRD structure revealsthat this material is also isostructural to the PC16 and PC14 monohydrateswith the same two crystallographically independent conformations andwater playing the same head group spanning role 3b, with 26 There are no further changes shown by DSC or TGA untila melt-decomposition endotherm forms, onset 265.4 °C, in agreementwith a previous report.27 Dehydration wasmonitored by XRPD. A sample of miltefosine monohydrate was placedon a watch glass and exposed to 120 °C in an oven for 12–120h. The XRPD pattern at each interval shows a reduction in crystallinityevidenced by considerable peak broadening . While an additional broad, low-angle peak appears at 2.6°2θ from 24 h drying onward, which might indicate the formationof a material with a larger unit cell, samples exposed proved to besticky with a tendency to agglomerate, indicating the dried materialis highly hygroscopic. These dehydrated samples were analyzed by DSC. The dehydrated samples exhibitevidence for a glass transition at about 55 °C, followed in somecases by a crystallization exotherm. All samples showed two furtherbroad endotherms at lower temperature than the original monohydrateimplying desolvation and melt-decomposition of the material of lowercrystallinity. This study reveals that after dehydration, the materialbecomes predominantly amorphous and implies that the water moleculeswithin PC16 hydrate are an integral structural feature of the crystalpacking arrangement. This is likely to arise from the lack of hydrogen-bonddonors in PC16 itself and hence the inability of the structure tostabilize the polar phosphate groups and span the breadth of the interdigitatedbilayer arrangement.In an attempt to find an anhydrousform of PC16, a range of dehydration, recrystallization, and desolvationstudies were undertaken under various conditions. The results weremonitored by X-ray powder diffraction (XRPD) and differential scanningcalorimetry (DSC). The DSC thermogram of miltefosine monohydrate showsa dehydration endotherm with an onset temperature of 97.8 °C,accompanied by a mass loss of 3.43% by TGA at the same temperaturecorresponding to 0.8 water molecules for formula unit. This substoichiometricvalue may represent some empty sites in the crystals.Powdered PC16 hydrate was recrystallizedfrom both water and methanol and analyzed by XRPD and DSC. A comparisonof the XRPD patterns of the calculated and experimental powder patternsfor the resulting PC16 hydrate and methanolate is shown in The PC16 samples recrystallized from water and methanol were thendesolvated by being kept in an oven for 24 h at 60 °C. The XRPDpatterns reveal no change in the case of the monohydrate and transformationof the methanolate to the monohydrate structure by adsorption of atmosphericmoisture during sample handling.d-glycero-1-phosphocholine, even though the d-glycero substituent significantly alters the packing arrangement.21 The conformation of the head group of the PCn molecules is dictated by the hydrogen-bonding nature ofthe solvent, and the interdigitated bilayer structure is retainedthroughout. Each structure is a conformational isomorph with a Z′ value of 2. Dehydration studies of PC16 hydratereveal that the dehydrated material is of low crystallinity and isunstable and readily reforms the hydrate. Water or methanol acts asan integral part of the structure bridging the bilayer breadth andstabilizing the strong hydrogen-bond-acceptor phosphate groups.TheSC-XRD structures of PC16 and other biologically active analogues(PC14 and PC12) demonstrate their tendency to crystallize as an isostructuralseries of monohydrates or methanolates. Hydrate formation is alsoobserved in the case of the more bulky 3-octadecyl-2-methyl-–1 over a wavenumber region of 4000–500 cm–1. DSC studies were carried out using a NETZSCH DSC214 Polyma  operated with nitrogengas. Samples (approx. 4–6 mg) were weighed in an aluminum panand hermetically sealed, and the lid was pierced. Samples were thenheated at 20 °C min–1 from 20 to 300 °C.TGA was carried out by Ruston Services using a TA Instruments Q 500TGA analyzer. Between 1 and 5 mg of the sample was weighed in platinumpans, and dry nitrogen was used as the purge gas (flow rate: 60 mLmin–1). XRPD patterns were recorded on glass slidesusing a Bruker AXS D8 ADVANCE diffractometer with a Lynxeye SollerPSD detector or a Bruker D2 phaser diffractometer equipped with aLYNXEYE XE-T detector using Cu Kα radiation at a wavelengthof 1.5406 Å. Single-crystal structures were collected at 120K using the Bruker D8 Venture diffractometers Photon III MM C14 orC7 CPAD detector, IμS- or IμS-III-microsource, focusingmirrors; λMoKα radiation (λ = 0.71073 Å) equippedwith Cryostream (Oxford Cryostreams) open-flow nitrogen cryostats.All structures were solved using direct methods and refined by full-matrixleast squares on F2 for all data usingSHELXL28 and OLEX229 software. All nondisordered nonhydrogen atoms were refined withanisotropic displacement parameters; disordered atoms in structuresPC14 (solvate) and PC16 (solvate) were refined with fixed equal occupancies.CH hydrogen atoms were placed in calculated positions and assignedan isotropic displacement factor that is a multiple of the parentcarbon atom and allowed to ride. H atoms attached to oxygen atomswere located on the difference map where possible or placed in calculatedpositions. Crystallographic data for the structures have been depositedwith the Cambridge Crystallographic Data Centre as supplementary publicationCCDC 2192420—2192424.All reagentsand solvents were purchased fromstandard commercial sources and used without further purification.FTIR was carried out using a PerkinElmer Spectrum 100 spectrometer,fitted with a diamond universal attenuated total reflectance accessory.Eight scans were collected for each sample at a resolution of 2 cmn-hexadecylphosphocholine,PC16) is a white crystalline powder supplied in its hydrated form.Analysis calcd for C21H48NO5P: C,59.26; H, 11.37; and N, 3.29%. Found: C, 58.98; H, 11.23; and N, 3.32%.Crystals of PC16 hydrate were obtained by dissolving PC16  in chloroform/toluene , shaking the sealedvessel, and sonicating at 70 °C for 1 min and cooling to roomtemperature, which yielded colorless birefringent block crystals after3 weeks. Crystal data: M = 425.57 g mol–1, triclinic, space group P1̅ (no. 2), a = 9.4657(7) Å, b = 10.8513(8) Å, c = 24.2121(18) Å, α = 90.460(2)°, β= 94.964(2)°, γ = 91.296(2)°, V =2476.9(3) Å3, Z = 4, Dc = 1.141 g/cm3, μ = 0.139 mm–1, F(000) = 944.0, 42928 reflections collected, 10780unique (Rint = 0.0692). Final GooF = 1.051, R1 = 0.0626 [7665 reflections with I ≥ 2σ(I)], wR2 = 0.1317 , 529 parameters, 0 restraints.Miltefosine  was combined with 2-butanol (0.30 mL),heated to 95 °C, sealed and shaken, cooled to room temperature,and reheated to 95 °C. The resulting colorless solution was leftto cool slowly, which yielded colorless needle crystals of a miltefosinemethanol solvate after 3 weeks. The sample was inadvertently exposedto methanol in the laboratory. Solvent molecules are disordered. Crystaldata: M = 447.61 g/mol, triclinic, space group P1̅ (no. 2), a = 8.6690(9) Å, b = 10.9861(13) Å, c = 27.648(3) Å,α = 95.027(5)°, β = 95.074(4)°, γ = 96.828(4)°, V = 2591.4(5) Å3, Z = 4, Dc = 1.147 g/cm3, μ = 0.137mm–1, F(000) = 994.0, 24643 reflectionscollected, 9812 unique (Rint = 0.1166).Final GooF = 1.032, R1 = 0.0976 [4625reflections with I ≥ 2σ(I)], wR2 = 0.2618 , 515 parameters,0 restraints.Miltefosine (n-Tetradecylphosphocholine(PC14) is a white crystalline powder and is supplied as a hydrate.Analysis calcd for C19H44NO5P: C,57.40; H, 11.16; and N, 3.52%. Found: C, 57.46; H, 11.03; and N, 3.45%.Acetonitrile (0.80 mL) was added to n-tetradecylphosphocholine, heated to 80 °C, sealed and shaken, cooledto room temperature, and then heated to 80 °C. The sealed vesselwas allowed to cool slowly, yielding colorless birefringent platecrystals of n-tetradecylphosphocholine monohydrateafter 1 week. Crystal data: M = 397.52 g/mol, triclinic,space group P1̅ (no. 2), a = 9.4389(12) Å, b = 10.7939(13) Å, c = 22.371(3) Å, α = 92.010(4)°, β= 90.886(4)°, γ = 91.099(4)°, V =2277.1(5) Å3, Z = 4, Dc = 1.160 g/cm3, μ = 0.147 mm–1, F(000) = 880.0, 44906 reflections collected, 9933unique (Rint = 0.1542). Final GooF = 1.007, R1 = 0.0737 [4573 reflections with I ≥ 2σ(I)], wR2 = 0.1914 , 484 parameters, 0 restraints.n-Tetradecylphosphocholine(PC14) methanol solvate was prepared as the result of a failed solutioncocrystallization of PC14 and t-butylhydroquinone in a 1:2 ratio,respectively. PC14 and t-butylhydroquinone were combined with acetonitrile(0.50 mL), heated to 80 °C, sealed, shaken, and cooled to roomtemperature. Methanol (0.10 mL) was then added, and the mixture washeated to 60 °C before sealing, shaking, and leaving to coolslowly. Colorless translucent prism-shaped crystals were yielded after4 weeks and were found to be a disordered methanol solvate of PC14in a 1:2.5 ratio. Crystal data: M = 418.55 g/mol,space group P1̅ (no. 2), a = 8.6781(6) Å, b = 10.9554(7) Å, c = 25.0960(16) Å, α = 99.656(2)°, β= 97.428(3)°, γ = 95.037(2)°, V =2317.7(3) Å3, Z = 4, Dc = 1.199 g/cm3, μ = 0.149 mm–1, F(000) = 926.0, 52057 reflections collected, 12261unique (Rint = 0.0492). Final GooF = 1.074, R1 = 0.1036 [9765 reflections with I ≥ 2σ(I)], wR2 = 0.2896, 658 parameters, 46 restraints.n-Dodecylphosphocholine(PC12) is a white crystalline powder and is supplied in its hydratedform. Analysis calcd for C17H40NO5P: C, 55.26; H, 10.91; and N, 3.79%. Found: C, 55.68; H, 10.80; andN, 3.67%. Acetonitrile (0.70 mL) was added to n-dodecylphosphocholine, heated to 80 °C, sealed and shaken, cooledto room temperature, and then heated to 80 °C. The sealed vesselwas allowed to cool slowly, yielding colorless birefringent platecrystals of n-dodecylphosphocholine monohydrate after5 days. Crystal data: M = 369.47 g/mol,, triclinic,space group P1̅ (no. 2), a = 9.4601(7) Å, b = 10.7497(7) Å, c = 20.9618(14) Å, α = 81.907(2)°, β= 86.909(3)°, γ = 89.774(2)°, V =2107.4(3) Å3, Z = 4, Dc = 1.165 g/cm3, μ = 0.154 mm–1, F(000) = 816.0, 40972 reflections collected, 11166unique (Rint = 0.0811). Final GooF = 1.032, R1 = 0.0730 [7489 reflections with I ≥ 2σ(I)], wR2 = 0.2012, 547 parameters, 0 restraints.


Scientific Reportshttps://doi.org/10.1038/s41598-019-50380-0, published online 27 September 2019Correction to: This article contains an error in Figure 5, where panel ‘PNA-30/HepG2 Day 7’ is a duplication of panel ‘PNA-0/HepG2 Day 14’, and ‘PNA-10/HepG2 Day 14’ is a duplication of ‘PNA-50/HepG2 Day 1’.The corrected Figure


The title compound consists of 1-butyl-3-methylimidazolium cations, tribromido(triphenylphophine)nickelate(II) anions and co-crystallized 1-butanol solvent molecules. 8H15N2)[NiBr3(P(C6H5)3)]·0.5C4H10O, was obtained in the form of single crystals directly from the reaction mixture. The molecular structure consists of separated 1-butyl-3-methylimidazolium cations, tribromido(triphenylphosphane)nickelate(II) anions and half a solvent molecule of 1-butanol, all connected via multiple hydrogen contacts to form a three-dimensional network. The co-crystallized 1-butanol molecule is disordered and adopts two orientations. The central C—C bonds of both orientations are located on an inversion centre (Wyckoff site 2b of space group P21/n). Thereby, each orientation has again two orientations with the OH group being located either on one or the other side of the C4 alkyl chain. The dried solvent-free compound exhibits a relatively low melting point (m.p. = 412 K).The solvated title salt, (C X3L]−  have been thoroughly characterized for more than 50 years by means of magnetic investigations, UV–vis and far-infrared spectroscopy, NMR and EPR as well as Mössbauer spectroscopy 3]− anion have been investigated as suitable precatalysts for the generation of Ni–NHC complexes (NHC: N-heterocyclic carbene) in catalytic processes, e.g. selective cross-coupling reactions [NiBr3P(C6H5)3] are known for the following cations: [As(C6H5)4]+ imidazolium; Xu et al., 2013+ 3)]−·0.5(C4H10O) (BMIm+ is 1-butyl-3-methylimidazolium).Nickel(II) complexes exhibiting pseudo-tetrahedral symmetry [Ni4 alkyl chain. All bond lengths and angles within the cation as well as the complex anion are in the expected ranges  to 117.94 (2)°, and Br—Ni—P angles ranging from 102.76 (2) to 106.26 (2)°. The packing of the molecular entities is depicted in Fig. 2The asymmetric unit of the title compound consists of one 1-butyl-3-methylimidazolium cation and one tribromido(triphenylphosphane)nickelate(II) anion Fig. 1. An addiet al., 20132  in 30 ml of 1-butanol. The resulting green precipitate was completely dissolved by heating up the suspension to the boiling point of the solvent. The hot solution was cooled down to 277 K overnight in a refrigerator. The resulting blue crystals were filtered off, washed thoroughly with diethyl ether and dried in vacuum at ambient conditions .The title compound was obtained as blue crystals in multi-gram scale from 1-butyl-3-methylimidazolium bromide, triphenylphosphane and anhydrous nickel(II) bromide in boiling 1-butanol : C 42.61 (44.62); H 4.28 (4.32); N 4.18 (4.00).Analytic data for Cb of space group P21/n. It is disordered with two different orientations, which were refined using a split arrangement with the sum of occupational factors being fixed to full occupation. This results in a total of half a molecule per formula unit. The H atoms of the disordered 1-butanol molecule including that attached to the O atom were calculated at idealized positions and refined using riding models.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S241431462100818X/wm4149sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S241431462100818X/wm4149Isup2.hklStructure factors: contains datablock(s) I. DOI: 2102393CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


HTT gene for which no therapies are available. HTT mutation causes protein misfolding and aggregation, preferentially affecting medium spiny neurons (MSNs) of the basal ganglia. Transcriptional perturbations in synaptic genes and neuroinflammation are key processes that precede MSN dysfunction and motor symptom onset. Understanding the interplay between these processes is crucial to develop effective therapeutic strategies to treat HD. We investigated the role of protein kinase CK2α’, a kinase upregulated in MSNs in HD and previously associated with Parkinson’s disease (PD), in the regulation of neuroinflammation and synaptic function in HD. We used the heterozygous knock-in zQ175 HD mouse model and compared that to zQ175 mice lacking one allele of CK2α’ (zQ175:CK2α’(±)). CK2α’ haploinsufficiency in zQ175 mice resulted in decreased levels of pro-inflammatory cytokines, HTT aggregation, astrogliosis and transcriptional alterations of synaptic genes related to glutamatergic signaling. zQ175:CK2α’(±) mice also presented increased frequency of striatal miniature excitatory postsynaptic currents (mEPSCs), an indicator of synaptic activity, and improved motor coordination compared to zQ175 mice. Neuropathological and phenotypic changes mediated by CK2α’ were connected to alpha-synuclein (α-syn) dysregulation and correlated with differences in α-syn serine 129 phosphorylation (pS129-α-syn), a post-translational modification involved in α-synucleinopathy and shown to be regulated by CK2 in PD. pS129-α-syn was increased in the nuclei of MSNs in zQ175 mice and in the striatum of patients with HD, and it decreased in zQ175:CK2α’(±) mice. Collectively, our data established a novel connection between CK2α’, neuroinflammation and synaptic gene dysregulation with synucleinopathy in HD and suggested common molecular mechanisms of neurodegeneration between HD and PD. Our results also support CK2α’ inhibition as a potential therapeutic strategy to modulate neuronal function and neuroprotection in HD.Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the The online version contains supplementary material available at 10.1186/s40478-022-01379-8. HTT) gene. This mutation results in progressive misfolding and aggregation of mutant HTT protein (mtHTT) and preferentially affects GABAergic medium spiny neurons (MSNs) in the striatum . a, Kruskal-Wallis test of module expressions between zQ175 mice and WT mice. b IPA canonical pathway analysis of module genes for module “Red”. c, Cook’s distance (DESeq2), which is a measure of how much a single sample is influencing the fitted coefficients for a gene, for all tested samples (HET = CK2α’(+/-), KI = zQ175, KIHET = zQ175:CK2α’(+/-)). d, Network visualization of top 15% connected genes for module genes of module “Greenyellow”. The size of the circles was scaled by the absolute value of the mean log2-fold change between zQ175 and zQ175:CK2α’(+/-) mice. e, MA-plot of differential gene expression between HD (zQ175) and WT mice. f, Venn Diagram of differentially expressed genes between HD (zQ175) and WT mice in comparison to Langfelder 2016 data. g, MA-plot of differential gene expression between zQ175:CK2α’(+/-) and WT mice. h, Venn Diagram of differentially expressed genes between zQ175, WT, zQ175:CK2α’(+/-) and CK2α’(+/-) mice. i, Driver factors of gene expression variance (genotype and/or gender) for the DGEs identified between zQ175 and zQ175:CK2α’(+/-) were evaluated using R package variance Partition.Additional file 9. Differential Gene Expression analyses across the four different genotypes WT, zQ175, CK2α’(+/-) and zQ175:CK2α’(+/-).Additional file 10. Alteration in CK2α’ expression changes cytokine profiles but not microglia. a, RT-qPCR analysis for CK2α’ and IL-6 in STHdhQ7/Q7 (control) and STHdhQ111/Q111 (HD) cell (n = 5 independent experiments). Data were normalized to GAPDH and control cells. b, siRNA knockdown of CK2α’ for 24 h in STHdhQ111/Q111 cells and RT-qPCR. Data were normalized with GAPDH and relativized to non-targeting control siRNA-treated cells (scramble), (n = 6 independent experiments). c, d, Representative images of mouse cytokine array panels from striatum extracts of WT, zQ175, and zQ175:CK2α’(+/-) at 12-14 months of age (n = 6 mice/genotype). e, f, Iba1 immunoblotting in the striatum of 12 months old WT, zQ175 and zQ175:CK2α’, quantification was measured by image analyses using Image J software, GAPDH is used as a loading marker. g-i, Representative images show the labeling of Iba1 in the dorsal striatum of WT, zQ175 and zQ175:CK2α’(+/-) at 3, 6 and 12 months old (g). Images were analyzed using the Image J software. Scale bar, 100 µm. Iba1+ cell body area in 12 months old mice  (h) and percent of Iba1+ cells in 300mm2  (i). Error bars denote mean ± SEM, values were analyzed by Student's t test in d, and one-way ANOVA and Tukey post-hoc test in a, b, f, h, i. *p represent p-values comparing zQ175 and WT, #p are p-values comparing zQ175 and zQ175:CK2α’(+/-).Additional file 11. Expression analyses for astrocyte markers of A1, A2 and pan-reactive ) in WT, zQ175, CK2α’(+/-) and zQ175:CK2α’(+/-).Additional file 12. Expression analyses for microglia markers of A1 inducing and reactive microglia ) in WT, zQ175, CK2α’(+/-) and zQ175:CK2α’(+/-).Additional file 13. Decreased CK2α’ ameliorated astrogliosis in zQ175 mice. a, b, Representative images show the labeling (a) and quantification (b) of Glutamine synthetase  and CK2α’ of striatum sections from 12-month-old mice in WT, zQ175 and zQ175:CK2α’(+/-) . DAPI is used for nuclear staining. Scale bars, 50 µm. c, Coronal images of brain scans in 9.4T magnet showing the striatum voxel (green box) for MRS acquisition from each genotype at 22 months. d, Localized proton magnetic resonance spectra  obtained at 22 months of age from the striatum of WT, zQ175, and zQ175:CK2α’(+/-) mice ). Differences in myo-inositol (Ins) between WT and zQ175 mice and between zQ175 and zQ175:CK2α’(+/-) mice are shown with arrows. e, f, Mean concentrations of myo-inositol (Ins) (e) and other reliably quantified metabolites (f) in the striatum of WT (black), zQ175 (red), and zQ175:CK2α’(+/-) (gray) mice. Asc: Ascorbate/vitamin C; tCr: total creatine + phosphocreatine; GABA: gamma-aminobutyric acid; Glc: glucose; Gln: glutamine; Glu: glutamate; tCho: total phosphocholine + glycerophosphocholine; GSH: glutathione; lns: myo-inositol; Lac: lactate; NAA: N-acetylaspartate; NAAG: N-acetylaspartylglutamate; PE: phosphoethanolamine; Tau: taurine. Error bars denote mean ± SEM, values were analyzed by one-way ANOVA with Tukey’s post-hoc test. p-values < 0.05 are indicated.Additional file 14. Marker genes and their mean log2 fold change between zQ175 and zQ175:CK2α’(+/-) mice compared to WT.Additional file 15. SNCA regulates the expression of synaptic genes identified in IPA analysis in R6/1 mice. a, IB for α-syn (4D6 antibody) and mtHTT (EM48 antibody) in the striatum of 5 months old WT, SNCAKO, R6/1 and R6/1:SNCAKO (n=3 mice/genotype). b, RT-qPCR analyses for SNCA and genes identified in the IPA analyses to be connected to SNCA;  (n = 4-5 mice/genotype). Error bars represent mean ± SEM. Statistical analyses were conducted by Student’s t-test. p-values <0.05 are indicated. We also indicated p-values <0.09 for those genes that showed a trend toward decreased expression.Additional file 16. CK2a’ haploinsufficiency decreased the levels of pS129-α-syn. a, Representative pS129-α-syn IF images (D1R1R antibody) in the dorsal striatum of 12 months old WT, zQ175 and zQ175:CK2α’(+/-), Scale bar, 20 μm. b, pS129-α-syn and CK2α’ immunoblotting of striatum samples from 12-month-old zQ175 and zQ175:CK2α’(+/-) mice (n = 3 mice/group). c, Levels of pS129-α-syn and CK2α’ were calculated using Image J from immunoblotting images in a and showed a parallel decrease of pS129-α-syn and CK2α’ levels in zQ175:CK2α’(+/-) compared to zQ175 mice. Error bars represent mean ± SEM. Statistical analyses were conducted by Student’s t-test. p-values <0.05 are indicated (n=3 mice/genotype).Additional file 17. Video showing pS129-α-syn nuclear localization (red) in the striatum of 12-month-old zQ175 mice. DAPI (blue) stains nuclei.Additional file 18. Supplementary Methods.


In clinical practice, this may result in an ineffective therapeutic regimen and, consequently, in treatment failure. Here, we report the crystal structures of IMP-6 and IMP-1 with the same space group and similar cell constants at resolutions of 1.70 and 1.94 Å, respectively. The overall structures of IMP-6 and IMP-1 are similar. However, the loop region (residues 60–66), which participates in substrate binding, is more flexible in IMP-6 than in IMP-1. This difference in flexibility determines the substrate specificity of IMP-type metallo-β-lactamases for imipenem and meropenem. The amino acid at position 262 alters the mobility of His263; this affects the flexibility of the loop via a hydrogen bond with Pro68, which plays the role of a hinge in IMP-type metallo-β-lactamases. The substitution of Pro68 with a glycine elicited an increase in the Km of IMP-6 for imipenem, whereas the affinity for meropenem remained unchanged.IMP-type metallo-β-lactamases confer resistance to carbapenems and a broad spectrum of β-lactam antibiotics. IMP-6 and IMP-1 differ by only a point mutation: Ser262 in IMP-1 and Gly262 in IMP-6. The   BcIIBacillus cereus metallo-β-lactamaseGESGuiana extended-spectrum β-lactamaseIMPimipenemaseKPCKlebsiella pneumoniae carbapenemaseMBLmetallo-β-lactamaseNDMNew Delhi metallo-β-lactamaseOXAoxacillin resistant β-lactamaseVIMVerona Integron-encoded metallo-β-lactamase
© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Received July 12, 2022; accepted September 24, 2022; published online September 20, 2022
 Enterobacteriaceae have aroused a growing concern  . In Japan, clinical isolates of carbapenemase-producing Enterobacteriaceae mainly produce IMP-type carbapenemases (EC 3.5.2.6), especially IMP-1 and IMP-6   and its complex with a mercaptocarboxylate inhibitor (PDB ID 1DD6) in 2000  (S. marcescens IMP-1 (PDB ID 5EV6) shows both open/closed structures of the mobile loop (L1) in different molecules in the asymmetric unit (P. aeruginosa IMP-13 show the mobile loop (L1) in both the open (PDB ID 6R79) and closed (PDB ID 6R78) conformations . The blaIMP-1 and blaIMP-6 genes were amplified by PCR using primers IMP-6-full-F (5′-AGCAAGTTATCTGTATATATTTTTGTTTTG-3′) and IMP-6-mat-R-Bam (5′-ATATAGGATCCTTAGTGGTTTTGATGGTTT-3′). PCR products were digested with the restriction enzyme Bam HI . A pET28a vector  was digested with Nco I, blunted with a blunting kit  and further digested with Bam HI. The vector and the inserts blaIMP-6 and blaIMP-1 were ligated using T4 ligase to generate the pET28a-imp6 and pET28a-imp1 plasmids, respectively. Escherichia coli BL21(DE3) cells were transformed with each plasmid.The starting plasmids were obtained from the clinical isolates Escherichia coli transformants carrying blaIMP-6 or blaIMP-1 were inoculated into a Luria-Bertani medium supplemented with 30 μg ml−1 kanamycin and incubated at 310 K. Isopropyl thio-β-d-galactoside  was added when the optical density at 600 nm reached 0.5, and the culture was further incubated for 20 h at 293 K. Cells were resuspended in buffer A (20 mM Tris/HCl (pH 7.5), 50 μM ZnSO4) and sonicated. The cell-free extract was applied to a Macro-Prep High S Support column  and washed with buffer A. The enzymes were eluted with buffer A containing 0.3 M NaCl. Fractions containing IMP-6 or IMP-1 were pooled, dialysed against buffer A and injected into a CM-Toyopearl 650S column  equilibrated with buffer A. The enzymes were eluted with a linear gradient of NaCl (0–0.25 M) in the same buffer. Analysis of the N-terminal amino acid sequence of IMP-6 confirmed that the signal sequence was removed in the host cells.4 and concentrated to 15 mg ml−1. IMP-6 drops were prepared by adding 3 μl protein solution and 3 μl reservoir solution containing 0.1 M sodium acetate (pH 4.6), 0.2 M ammonium acetate and 17% (w/v) polyethylene glycol 8000; these drops were equilibrated over 0.5 ml reservoir solution using the hanging-drop vapour-diffusion method at 288 K. IMP-1 drops were prepared similarly, but the reservoir solution contained 0.1 M sodium acetate (pH 5.0), 0.2 M ammonium acetate and 22% (w/v) polyethylene glycol 8000. Rod-shaped crystals grew to a maximum dimension of 0.1 × 0.1 × 0.2 mm in 2 weeks. IMP-6 and IMP-1 crystals were flash-frozen in liquid nitrogen using the respective reservoir solution containing 10% (v/v) glycerol as a cryoprotectant. X-ray diffraction data were collected at 100 K on beamline BL44XU at SPring-8  using an Eiger X 16 M detector . Data were processed using XDS  with 50 μM ZnSOP. aeruginosa IMP-1 (PDB ID 1DDK) as the search model. Manual model building and refinement were carried out using Coot (The structures were solved by the molecular replacement using the Molrep (IMP-13 in complex with hydrolysed imipenem (PDB ID 6RZS) or IMP-13 in complex with hydrolysed meropenem (PDB ID 6RZR) was superposed on IMP-6. Docking simulations between IMP-6 and hydrolysed substrates were performed using the MF myPresto ver. 3.2 mmMPApp2 application .The P68G mutants of IMP-6 and IMP-1 were constructed by site-directed mutagenesis using a KOD plus mutagenesis kit . Plasmids pET28a-imp6 or pET28a-imp1were used as templates. The following oligonucleotide primers were used: P50Gfwd (5′-GGGTGGGGCGTTGTTGGTAAACATGGTTTGGTGG-3′) and P50Grev (5′-CCACCAAACCATGTTTACCAACAACGCCCCACCC-3′). The introduced mutation was confirmed by DNA sequencing. The mutant enzymes were expressed and purified using the same method as that of the wild-type enzymes.4. The wavelengths and extinction coefficients used in this study were the same as those reported by Laraki et al.  to prevent denaturation. The values of the kinetic parameters (mK and kcat) were obtained by a double-reciprocal (Lineweaver-Burk) plot of initial steady-state velocities at different substrate concentrations  at 303 K in 20 mM HEPES buffer (pH 7.0) with 50 μM ZnSOP. aeruginosa IMP-1 as the molecular replacement template. The structure of IMP-6 was refined to a crystallographic R-factor of 20.8% (Rfree = 23.3%) for 112,203 unique reflections in the resolution range from 48.3 to 1.70 Å. The structure of IMP-1 was refined to a crystallographic R-factor of 25.8% (Rfree = 28.7%) for 75,259 unique reflections in the resolution range from 46.1 to 1.94 Å. Rmerge of IMP-1 are worse than those of IMP-6  for the main-chain atoms between the chains A, B, C and D of IMP-6 were all less than 0.50 Å, indicating that the four protein molecules are nearly identical . AlthougS. marcescens IMP-1 . Zn1 is tetrahedrally coordinated with His116, His118, His196 and a water molecule (wat237); Zn2 is coordinated with Asp120, Cys221, His263 and wat237. The coordination geometry of two zinc ions in IMP-6 is nearly identical to that reported for IMP-1  and S. marcesces IMP-1 (PDB ID 5EV6) have been reported as IMP-1 from other species. The 1DDK structure is difficult to compare because of its low resolution and data quality. In the case of 5EV6, the average B factor of the loop region was 33.892 (x) while that of the non-loop region was 33.488 (y), with a ratio of 1.01 (x/y). This value is inconsistent with our results.The L1 loop, also termed the flap region ,,. TherefoKm values have shown that IMP-2 and IMP-13 have a high affinity for meropenem, whereas IMP-18 showed a high affinity for imipenem . The main chain RMSD for both is 0.214, so there is no possibility of conformational change even under neutral conditions. In the case of IMP-1, Hinchliffe et al. previously submitted a crystal structure at pH 6.0 , imipenem is in three different conformations. This may be due to the weaker π–sulphur interaction with the side chain of Trp64 and the weaker hydrophobic interaction compared to meropenem  from the Japan Society for the Promotion of Sciences.None declared.The coordinate and structure factor files from this study have been deposited in the Protein Data Bank under the accession numbers 7XHW (IMP-1) and 7XHX (IMP-6).