Penicillium contamination in crops/foods. The antifungal efficacy of hydrogen peroxide  was investigated in Penicillium strains by using kojic acid (KA) as a chemosensitizing agent, which can enhance the susceptibility of pathogens to antifungal agents. Co-application of KA with H2O2 (chemosensitization) resulted in the enhancement of antifungal activity of either compound, when compared to the independent application of each agent alone. Of note, heat enhanced the activity of H2O2 to a greater extent during chemosensitization, whereby the minimum inhibitory or minimum fungicidal concentrations of H2O2 was decreased up to 4 or 13 fold, respectively, at 35–45 °C (heat), when compared to that at 28 °C . However, heat didn’t increase the antifungal activity of KA, indicating specificity exists between heat and types of antifungals applied. The effect of chemosensitization was also strain-specific, where P. expansum  or P. italicum 983 exhibited relatively higher susceptibility to the chemosensitization, comparing to other Penicillium strains tested. Collectively, chemosensitization can serve as a potent antifungal strategy to lower effective dosages of toxic antifungal substances, such as H2O2. This can lead to coincidental lowering of environmental and health risks.Oxidative treatment is one of the strategies for preventing  H-pyran-4-one, KA, Aspergillus, Penicillium) or Acetobacter , as an antitumor or anti-leishmanial agent, etc. [Cryptococcus neoformans, a causative agent triggering human cryptococcosis, in which KA inhibited melanin biosynthesis required for fungal infectivity [Kojic acid (5-hydroxy-2-(hydroxymethyl)-4obacter . Recently, the use of atoxigenic  Aspergillus strains as biocontrol agents in agricultural environments [Certain ronments  has promronments , which cronments . Thereforonments .Penicillium are frequently involved in food contamination or postharvest decay. For example, P. expansum is the main producer of the mycotoxin patulin (PAT) that triggers harmful effects on human/animal health [Penicillium strains, such as P.digitatum and P. italicum are the most devastating citrus pathogens, causing significant economic losses to the industry during post-harvest [The filamentous fungi in the genus l health . Apples l health . Meanwhi-harvest . Therefoetc., can be sources of “abiotic” stress to fungal pathogens. Accordingly, abiotic stresses artificially applied could adversely affect the growth of fungi, resulting in the reduction of fungal contamination or postharvest decay. Heat or oxidative treatment is one of the strategies to prevent contamination by Penicillium in foods or crops. For example, immersion of pear fruit in heated water (55 °C) controlled P. expansum-triggered fruit decay, which also allowed the delay of fruit ripening during storage [2O2 and sodium hypochlorite (NaClO), resulted in synergistic antifungal effect on P. expansum, rendering effective control of postharvest decay in fresh fruits [Various environmental factors, such as fluctuation of temperature, humidity or pH,  storage . Likewish fruits .etc., increased the susceptibility of the yeast pathogen Candida albicans to FLC [Co-application of certain types of compounds with conventional antimicrobial drugs/fungicides can enhance the effectiveness of drugs/fungicides through a mechanism termed “chemosensitization” ,19,20,21s to FLC ,23,24.Penicillium  to high temperatures , are determined and compared according to the method outlined by the Clinical Laboratory Standards Institute (CLSI) M38-A [Penicillium strains to H2O2, lowering the effective dosages of H2O2 required for control of Penicillium. The potency of H2O2 is greater when Penicillium strains are treated with heat (35–45 °C).In this study, antifungal chemosensitization is investigated for effective control of pathogenic strains of lium See  by co-apI) M38-A . ResultsPenicillium was tested using P. expansum W1  as a representative strain. P. expansum W1 was cultivated on potato dextrose agar (PDA) at high temperatures  , and was then transferred to 28 °C  for growth recovery  on PDA when it was cultivated solely at 35, 45 or 55 °C for 7 d. However, P. expansum W1 treated with heat  for 1–4 d could recover growth after cells were transferred to 28 °C (grown up to 7 d). W1 cultivated at 55 °C could not recover growth even after 1 day-heat treatment at 55 °C. Based on this result, moderate (28 °C) to high  temperatures, except 55 °C, were chosen for further antifungal investigation in this study.As shown in Penicillium strains didn’t visibly grow in RPMI liquid medium (microtiter plates), and hence, the values of Minimum Inhibitory Concentrations (MICs)/Fractional Inhibitory Concentration Indices (FICIs) could not be determined . The values of FFCIs ranged from “additive” (0.5 < FFCI ≤ 1) to “neutral” (1 < FFCI ≤ 2) [2O2 and also KA in most Penicillium strains examined at 24 h, which was reflected in lowered MFCs of each compound when combined.At 24 h of CLSI-based fungal cultivation , termined , whereastermined  except PFCI ≤ 2)  . For example, the average MFCs (MFCAlone) of H2O2 in Group A were 7.0, 2.0 or 0.6 mM at 28, 35 or 45 °C, respectively, thus exhibiting the highest or lowest MFCAlone value at 28 or 45 °C, respectively. When H2O2 was co-applied with KA (chemosensitization), the average MFCs of H2O2 were lowered further, resulting in MFCCombined values of 4.0, 1.0 or 0.3 mM at 28, 35 or 45 °C, respectively. Therefore, the values of MFCCombined (chemosensitization) were 1.7 to 2.0 times lower comparing to that of MFCAlone (independent application). Of note, there was up to 13-fold decrease in MFCCombined in Group A at 45 °C (MFC: 0.3 mM) comparing to that at 28 °C (MFC: 4.0 mM).In general, the higher the temperature, the lower the concentration of H2O2 with KA lowered the MFC values of H2O2. Consequently, chemosensitization resulted in 1.2 to 1.8 times lower MFC values (MFCCombined) of H2O2 comparing to the independent application of H2O2, alone. The only exception is P. griseofulvum 2159, which showed indifference to chemosensitization . Comparing to Group A, the average MFCs of H2O2 for Group B were relatively higher, viz. 1.8 to 4.0 times higher for MFCAlone and 2.0 to 4.7 times higher for MFCCombined, respectively . Therefore, results indicated that Group B strains were less susceptible to H2O2 than the Group A.The effect of chemosensitization was also identified in Group B, where co-application of Hectively . Therefo2O2/KA. The MFC values of H2O2 in P. italicum 983 were 2, 1 or 0.125 mM for MFCAlone (independent application) and 1, 1 or 0.0625 mM for MFCCombined (chemosensitization) at 28, 35 or 45 °C, respectively  heat enhanced H2O2 activity and (2) chemosensitization enhanced the antifungal activity of H2O2 (Exception: no change in MFCCombined at 35 °C). However, tests with P. digitatum 786, the other citrus pathogen, showed that while the MFCs (MFCAlone) of H2O2 were lowered from 4 to 1 mM when temperature was increased from 28 to 35 °C , this strain was unable to grow at 45 °C . Moreover, P. digitatum 786 showed indifference to KA-mediated chemosensitization, resulting in no change in MFC values when KA was co-applied with H2O2  enhanced the activity of H2O2, while that of KA was vastly unaffected by heat treatment. Regarding the stability of H2O2, H2O2 is sensitive to light, pH and/or heat. Especially, heat can induce chemical decomposition of H2O2 into H2O and O2. The O2 generated can be used for mitochondrial respiration in fungi, while it also means increase of pressure. Therefore, O2 level and pressure as well as the level of mitochondrial respiration may be of interest in the future chemosensitization study.Altogether, at 24 h, KA chemosensitizes most Penicillium strains or growth temperatures. For instance, MICs/FICIs could be measured in Group A or C at 28 °C (in microtiter plates), while those values could not be determined at 35 or 45 °C due to incapability of their growth at the higher temperatures. Whereas, MICs/FICIs could be determined in Group B at both 28 and 35 °C (45 °C: no growth), thus showing relatively higher tolerance of Group B to heat (35 °C) comparing to Group A or C in the liquid culture  could be determined depending on types of  culture .Alone or MICCombined, of H2O2 or KA were lowered after chemosensitization, as follows: (1) 2.2 or 3.5 times lowered for H2O2 or KA, respectively, in Group A (28 °C), (2) 1.5 or 1.7 times lowered for H2O2 and 1.4 or 2.1 times lowered for KA at 28 or 35 °C, respectively, in Group B, and (3) 2.0 times lowered for both H2O2 and KA in Group C (28 °C). In all Groups, FICI values ranged from “additive” (0.5 < FICI ≤ 1) to “neutral” (1 < FICI ≤ 2) [Combined when temperature was increased from 28 °C (MIC: 5.3 mM) to 35 °C (MIC: 1.3 mM). Despite the absence of calculated “synergistic” interaction, as determined by “additive” or “neutral” interactions  . Of noteractions , there wP. glabrum 766, P. chrysogenum 824, P. italicum 983 and P. digitatum 786, which were unable to grow at 45 °C. Noteworthy is that, when compared to 24 h  for 48 h, ≥99.9% fungal death was achieved with 2.0, 0.5 or 0.2 mM of H2O2 at 28, 35 or 45 °C, respectively .The values of MFCs/FFCIs could also be determined on PDA for most strains  at 28, 35 and 45 °C. Exceptions are ectively , while s at 24 h . Therefo2O2 needed for achieving ≥99.9% killing of Penicillium strains at 48 h. For example, the average MFCAlone values of H2O2 in Group A at 48 h were 4.0, 1.5 or 0.4 mM at 28, 35 or 45 °C, respectively, indicating 10 times lower concentration of H2O2 (45 °C) was required to achieve ≥99.9% fungal death, when compared to 28 °C. With the chemosensitization, MFCCombined values of H2O2 were lowered further to 2.0, 0.5 or 0.2 mM at 28, 35 or 45 °C, respectively, thus showing that 10 times less H2O2 was needed to achieve ≥99.9% fungal death at 45 °C, when compared to 28 °C. Similar trends in the heat enhancement of H2O2 activity were also found in Groups B and C during chemosensitization. As observed in 24 h, the average MFCs of H2O2 in Group B (both MFCAlone and MFCCombined) were relatively higher than that in Group A , thus confirming further that Group B was less susceptible to H2O2 than Group A.As observed in 24 h, the higher the temperature, the lower the concentration of HP. glabrum 766 and P.chrysogenum 824, were unable to recover their growth on PDA at the elevated temperature, viz. 45 °C (thus cannot determine their MFCs/FFCIs). However, Group A strains could still recover their growth at 45 °C. Thus, results indicated differential range of optimum growth temperatures for each fungus tested.Interestingly, although Group B exhibited higher tolerance to heat (at 35 °C) comparing to Group A or C , two strains in Group B, 2O2, where the level of MFCCombined of KA (chemosensitization) was 1.3 to 2.4 times lower than MFCAlone (independent application), depending on types of strains  enhanced the activity of H2O2, while that of KA was vastly unaffected by heat treatment. Therefore, results proved further that specificity exists between heat and types of antifungals applied. Also, effect of chemosensitization was strain-specific, where P. expansum  or P. italicum 983 exhibited relatively higher susceptibility to H2O2, comparing to other Penicillium strains. Considering KA can be oxidized by H2O2 under heating conditions, the oxidized KA might also possess an antifungal property. Determination of the precise amount of KA by using HPLC, etc., warrants future study.Collectively, at 48 h, KA chemosensitizes most P. expansum FR2 showed hypersensitivity to heat (45 °C) comparing to other P. expansum strains . As shown in P. expansum FR2 barely grew at 45 °C, where very tiny colonies appeared on PDA even without H2O2 or KA treatment. If FR2 was treated with H2O2 (as low as 0.25 mM) at 45 °C, colony growth was completely inhibited . Whereas, the impact of KA on the growth of FR2 at 45 °C was negligible  , even at the normal growth temperature (28 °C). Moreover, the color of FR3 colonies remained pale at all conditions, while that of W2 was blue, indicating cellular physiology or a process, such as secondary metabolism responsible for pigment development, in FR3 is also impaired. Similar type of interrelationship between fungicide resistance and reduced fungal growth was previously identified in other fungi [The other fludioxonil-resistant mutant, d in FR2 . However strain) a. For into 45 °C a. Howeveer fungi .etc., as determined in this study. Precise characterization of the links between heat susceptibility/reduced growth and fludioxonil resistance, identified in FR2 and FR3, warrants future study. Summary of agent interactions, between biotic and abiotic stressors, is described in Altogether, results from two fludioxonil resistant mutants  showed that one or more traits are co-segregated with fungicide resistance. Previous study showed that certain fungi with mutations in genes responsible for signal transduction of environmental stress, such as Mitogen-Activated Protein Kinase (MAPK) signaling pathway, could develop fludioxonil resistance . CoincidPenicillium strains were cultured on potato dextrose agar (PDA) at 28 °C, except when otherwise noted in the text .Fungal strains used in this study are summarized in 2O2)) were procured from Sigma Co. . KA was dissolved in dimethylsulfoxide  before incorporation into culture media. Throughout this study, control plates (No treatment) contained DMSO at levels equivalent to that of cohorts receiving antifungal agents, within the same set of experiments. H2O2 was diluted in sterile water before incorporation into culture media.Antifungal compounds (kojic acid (KA) , hydrogePenicillium expansum W1 to high temperatures. First, fungal conidia (5 × 103) were spotted on PDA (triplicate PDA plates per each temperature), and were initially incubated at three different temperatures . Triplicate PDA plates were then removed from each temperature  at day 1, 2, 3, and 4, and were transferred to 28 °C for additional 6, 5, 4, and 3 d of growth, respectively, resulting in a total of 7 d of incubation for each treatment . For controls, Penicillium strains were grown solely at 28 °C or respective temperature  for 7 d. The level of growth recovery at 28 °C was evaluated based on fungal radial growth, which was compared to that determined at 28 °C.Agar plate-based bioassay was performed to evaluate susceptibility of 2O2  in the strains of Penicillium, checkerboard bioassays (triplicate) (0.4 × 104–5 × 104 CFU/mL) were performed in microtiter wells  using a broth microdilution method , according to protocols outlined by the Clinical and Laboratory Standards Institute (CLSI) M38-A [2O2 and KA) were defined as: synergistic (FICI ≤ 0.5), additive (0.5 < FICI ≤ 1), neutral (1 < FICI ≤ 2) or antagonistic (2 < FICI) [To determine the precise level of chemosensitizing activity of KA  to H < FICI) . If pref < FICI)  methodolt-test) was performed based on “Statistics to use” [p < 0.05 was considered significant.Statistical analysis  stressors were determined for the enhancement of antifungal efficacy as follows: With chemosensitization (KA + H2O2), (1) MIC or MFC of KA or H2O2 was lowered during chemosensitization depending on types of strains or culture conditions  may be the possible mechanism of enhanced activity of the combinational treatment (KA + H2O2).In this study, levels of interactions between biotic (KA) and abiotic (HP. expansum strains (Group A) tested were sensitive to KA-mediated chemosensitization. In Group B, P. glabrum 766 was sensitive to chemosensitization in almost all conditions tested, while P. griseofulvum 2159 was mostly insensitive to chemosensitization. P. chrysogenum 824 was sensitive to chemosensitization at 24 h, while it was insensitive at 48 h; (3) In Group C, the citrus pathogens, the effect of chemosensitization was dependent upon cultivation time and temperatures, where P. digitatum 786 showed less sensitivity to the chemosensitization comparing to P. italicum 983. Altogether, the effectiveness of KA-mediated chemosensitization with H2O2 was fungal strain-specific. We speculate that different levels of antioxidant defense efficiency, metabolism or responses to nitrosative stress, etc., in different Penicillium strains  may trigger the differential responsiveness of fungi to the chemosensitization. The fact that KA is produced by different Penicillium strains  Comparing to 24 h, 48 h of cultivation of Penicillium strains, in general, required lower concentration of H2O2 to achieve ≥99% fungal death. Results indicated that H2O2 effectively damaged the cellular integrity of Penicillium strains, which may result in the inhibition of cell division cycle as well as antioxidant defense and metabolism in fungi.Considering KA affects the cellular nitric oxide metabolism , it is p2O2 required for achieving ≥99.9% fungal death, indicating heat and H2O2 synergize each other for the enhancement of antifungal activity. However, heat did not enhance the activity of KA, indicating specificity also exists between heat and types of antifungal compounds applied for the antifungal efficacy; (2) P. expansum FR2, fludioxonil-resistant mutant, showed hypersensitivity to heat (45 °C), while the colony growth of P. expansum FR3, the other fludioxonil-resistant mutant, was severely reduced comparing to its parental strain at all temperatures tested. Results indicated that susceptible/sensitive targets for fungal control might be identifiable in such mutants, which is coincided with the mutation involved in fungicide resistance.The effect of temperatures during chemosensitization is determined as follows: (1) The higher the temperature, the lower the concentration of HP. expansum strains or P. italicum 983. Chemosensitization can lower effective dosages of toxic antifungal substances, such as H2O2, leading to coincidental lowering of environmental and health risks. Antifungal efficacy of H2O2 was greater when Penicillium strains were treated with heat . Considering that much higher temperatures  are conventionally used for fungal control in crops, the temperatures defined as heat in this study , especially during chemosensitization, are markedly lower, and thus could reduce crop damage associated with heat treatment. The use of safe chemosensitizing agents, such as KA, that debilitate fungal pathogens may be a viable approach to control agro/food fungal pathogens.In conclusion, KA, a safe, natural compound, possesses a potential to serve as an antifungal chemosensitizing agent in combination with oxidative stressor(s). This potential appears to be greatest with


Plasmodium falciparum, NMT substrates are important in essential processes including parasite gliding motility and host cell invasion. Here, we generated parasites resistant to a particular NMT inhibitor series and show that resistance in an in vitro parasite growth assay is mediated by a single amino acid substitution in the NMT substrate-binding pocket. The basis of resistance was validated and analyzed with a structure-guided approach using crystallography, in combination with enzyme activity, stability, and surface plasmon resonance assays, allowing identification of another inhibitor series unaffected by this substitution. We suggest that resistance studies incorporated early in the drug development process help selection of drug combinations to impede rapid evolution of parasite resistance.The attachment of myristate to the N-terminal glycine of certain proteins is largely a co-translational modification catalyzed by N-myristoyltransferase (NMT), and involved in protein membrane-localization. Pathogen NMT is a validated therapeutic target in numerous infectious diseases including malaria. In   •P. falciparum NMT inhibitor IMP-1002Discovery of a mutant offering resistance against •Structural basis of the mechanism of resistance revealed by X-ray crystallography•Genetic and chemical validation of resistance using CRISPR-Cas9 and enzyme assays•Crystal structures of PvNMT[G386E] with IMP-1002 versus DDD85646, overcoming resistance Plasmodium falciparum combined with inhibitors decipher how a point mutation in nmt leads to the development of resistance against an inhibitor series, and enables identification of an NMT inhibitor that can overcome this resistance phenotype.Structural studies of N-myristoyltransferase (NMT) of the malaria parasite  Plasmodium, led to an estimated 216 million clinical cases and nearly half a million deaths in 2016  and fungi (nmt gene (PF3D7_1412800). Using coenzyme A (CoA)-activated lipid, NMT transfers myristate to the substrate protein, after removal of the initiator methionine, in a predominantly co-translational process  and P. vivax (Pv) NMTs has focused on generating potent compounds selective over the two human (Hs) NMTs known as HsNMT1 and HsNMT2, targeting the peptide-binding pocket, and optimizing the pharmacokinetic profile. Recent developments include exploitation of inhibitor scaffolds identified in two large high-throughput screens (Leishmania donovani (Ld) NMT as well as PfNMT, showing improved selectivity over HsNMT1 and HsNMT2 and with promising activity against liver-stage parasites  and variant in complex with inhibitor. Structure-guided analysis of these data enabled discovery of an inhibitor series that can overcome parasite resistance to NMTi. Collectively, our studies identify a mechanism of NMTi resistance early in the drug-development process, and provide a strategy to overcome parasite resistance to NMTi.P. falciparum mutant line resistant to NMTi IMP-1002 (50) inhibitory activity of 34 nM . This protein bridges the parasite plasma membrane and the IMC. GAP45 is anchored at the plasma membrane through myristoylation and palmitoylation of its N terminus and at the IMC through palmitoylation of its C terminus, and serves as a marker for correct IMC formation and recruitment of other proteins, such as MyoA and MTIP, to form the glideosome involved in invasion  was used to select for resistant parasites (50 for Dd2 parasites was comparable with that of the 3D7 line (50 in this parasite line) parasite growth was detected in one culture with an initial 106 parasite inoculum. Resistance to IMP-1002 was confirmed in the bulk population and three parasite clones , leading to an amino acid substitution from glycine to glutamic acid (G386E) in the NMT protein, was present in the bulk population and each individual clone.The arasites . IMP-1003D7 line A. After e clones B. Both I0 values B. To disnmt gene in 3D7 parasites. Genetic modification of P. falciparum 3D7  during the period 36–45 h post-invasion, when myristoylation is at its peak  as a tractable model for PfNMT. PvNMT possesses 80% sequence identity and 93% similarity to PfNMT and is an excellent platform for structural studies, while to date PfNMT has proven intractable for X-ray crystallography. Activity measurements with PvNMT[WT] and PvNMT[G386E] enzymes used MyrCoA and a 15-residue NMT substrate peptide. The enzymes were of similar purity (50) for PvNMT[G386E] compared with PvNMT[WT], which indicated a shift to higher concentration for the variant enzyme visualized through a ratio of G386E/WT of 2.6 on average  (6MAY) and that of PvNMT[WT] (PDB: 2YNC) (6MB1), Y211 moves to a previously unobserved third rotamer conformation (Y211c) to form a π-π stacking interaction with the pendant phenyl ring of IMP-1002, while H213 remains in the unliganded position H213a  . There iB: 2YNC)   A, with ton H213a B. On IMPon H213a B. Both Yon H213a B.2YND) , strengthening the hypothesis that DDD85646 binding is unaffected by G386E (m) between PvNMT[WT] and PvNMT[G386E] was only 3°C, significantly less than the >7.0°C in favor of WT observed for IMP-1002   revealed2YND) C. Therefby G386E  and S3. IMP-1002 A.6MAZ)  , and dis6MAZ) C. Analys6MAZ) D. By inc6MAZ) C, and thtability , and S5 2YNE) (4UFX) (50 shift assay with the parasite and IC50 determination with the CPM assay using IMP-0964 (a close derivative of IMP-0856) and IMP-0320. We also included the NMTi IMP-0917, a close derivative of IMP-1002 that has recently been reported   and IMP-) (4UFX)   (4UFX)  and S6B reported , which wreported . Shifts rted  to determine the relative KD values of different NMTi with the two forms of the enzyme. SPR improved differentiation between the binding affinities of NMTi for PvNMT[WT] and PvNMT[G386E], as summarized in To complement the CPM assay and potentially increase the accuracy of measuring ICD SPR analysis. In combination with the shift in EC50, IC50, and thermal stability, the SPR data clearly show that DDD85646 can overcome resistance mediated by G386E.The kinetic differences between the compounds with the WT and G386E enzymes reflected this generally weaker binding, with faster off rates apparent from the sensorgram traces. IMP-1002 and IMP-0917 exhibited interesting kinetics: both showed a slow dissociation rate with PvNMT[WT] C, suggesPlasmodium NMT, identified by drug selection of a non-synonymous point mutation in the nmt open reading frame. The importance of this mutation in P. falciparum parasites for this drug target, was confirmed by CRISPR-Cas9-mediated substitution of this single nucleotide in the WT parasite, recapitulating resistance against three chemically distinct NMT inhibitor series. Data from the PfNMT[G386E]-resistant parasite line were used to guide biochemical and structural analysis of this variant in recombinant PvNMT, providing both enzymatic inhibition data and the crystal structures of the WT and G386E variant with bound MyrCoA and the NMT inhibitor (NMTi) used for mutation selection. Structural, IC50 and KD data for PvNMT[G386E] are consistent and correlated with the EC50 shift data obtained in G386E parasites, although the active site of PvNMT differs from PfNMT in 2 out of 23 residues   in the substrate-binding pocket of o PvNMT) . These sInterestingly, the G386E variant parasite shows no apparent reduction of fitness in growth at the asexual blood stage, although it is currently unknown whether this observation would apply throughout the complete parasite life cycle. Even though G386E reduces the space in the peptide-binding pocket, the flexibility of Y211, E386, and H213 C providein vitro. Structural biology has been applied extensively to drug resistance in other fields including influenza  was dissolved in dioxane (5 mL) and treated with 4-fluoro-2-hydroxybenzene boronic acid  and tetrakis(triphenylphosphine) palladium(0) (20 mg), followed by a solution of potassium phosphate  in water (1 mL). The reaction mixture was heated under reflux for 2 h, cooled to room temperature and evaporated under reduced pressure. The residue was partitioned between EtOAc (20 mL) and saturated sodium bicarbonate solution (20 mL). The organic phase was dried over Na2SO4, concentrated under reduced pressure and the crude product purified by flash column chromatography by elution with DCM/EtOAc  to give 5-(4-fluoro-2-hydroxyphenyl)-1methyl-1H-indazole-3-carbaldehyde as a colourless solid ; mp 212-214°C. 1H NMR  10.15 , 8.30 , 7.73 , 7.69 , 7.32 , 6.74 – 6.64 , 4.62 , 4.24 .A solution of 5-iodo-1-methyl-1H-indazole-3-carbaldehyde  and triphenylphosphine  in THF (4 mL) was treated with a solution of 2-ethanol  in THF (3 mL). The reaction mixture was cooled to 0°C before being treated with di-isopropyl azodicarboxylate . The reaction mixture was allowed to warm to room temperature, stirred overnight and concentrated under reduced pressure. The crude product was purified by column chromatography by elution with hexane/IPA (gradient 80:20 to 70:30) to give a mixture of the desired product and unreacted alcohol. The crude product was further purified by elution with DMC/methanol (97:3) to give 5-ethoxy)phenyl)-1-methyl-1H-indazole-3-carbaldehyde as a colourless oil . 1H NMR  10.22 , 8.37 , 7.57 , 7.46 , 7.29 , 6.72 , 6.67 , 4.21 , 3.94 , 3.62 , 2.74 , 2.04 , 1.95 .A solution of 5-(4-fluoro-2-hydroxyphenyl)-1-methyl-12SO4) and concentrated under reduced pressure. The crude product was purified by column chromatography by elution with EtOAc/diethylamine (95:5) to provide 1-ethoxy)phenyl)-1-methyl-1H-indazol-3-yl)-N,N-dimethylmethanamine as a colourless oil . LC-MS Rt = 11.6min, MH+ 436; 1H NMR  7.88 , 7.48 , 7.35 , 7.33 – 7.26 , 6.73 , 6.67 , 4.08 , 3.93 , 3.83 , 3.63 , 2.75 , 2.33 , 2.08 , 1.92 ; 13C NMR  162.76 , 156.81 , 145.75, 142.38, 140.07, 136.91, 131.89 , 130.14, 128.66, 127.19, 123.46, 121.30, 111.62, 108.19, 107.20 , 100.39 , 68.63, 55.81, 45.59, 35.75, 35.41, 23.66, 11.71, 9.34; ESI HRMS, found 436.2510 .A solution of 5-ethoxy)phenyl)-1-methyl-1H-indazole-3-carbaldehyde  in THF (3 mL) was treated with a solution of dimethylamine in THF , followed by glacial acetic acid . The solution was stirred at room temperature for 10 min before addition of solid sodium triacetoxy-borohydride  and DCE (2 mL). The reaction mixture was stirred at room temperature for 18 h before being quenched with sodium carbonate solution . DCM (20 mL) was added and the layers were separated. The organic extract was dried  in PBS for 10–20 min, permeabilized in 0.1 % Triton X-100 in PBS for 10 min, and blocked with 3 % BSA in PBS for at least 30 min at 4°C. GAP45 detection was achieved using a primary rabbit anti-GAP45-antibody . Slides 50 determination in 3D7 in a final volume of 100 μL. For the EC50 of PfNMT[G386E] compared to PfNMT[WT] a range from 0.2 nM – 10 μM for IMP-1002 and DDD85646 and 0.6 nM - 40 μM for IMP-0964 and IMP-0320 was used. The final DMSO percentage of 0.08% did not interfere with parasite development determined by Giemsa-stained thin blood smears. Cultures were incubated in gassed humidity chambers  at 37°C for a further 96 h to increase the dynamic range and processed as described , with subtraction of the background signal from uninfected RBC. When comparing the EC50 of two parasite lines, Prism software was used to fit and constrain EC50 curves at the top and bottom to improve plateau definition through a shared value of all data sets.Aliquots of 100 μL ring-stage parasites (1–6 h post invasion [PI]) were diluted in 96-well flat bottom black plates to 0.3 % parasitemia and 2 % hematocrit. Inhibitors were added in doubling dilutions ranging from 7.8 nM – 5 μM for DDD85646 and 0.54 nM – 400 nM for IMP-1002 for the initial ECescribed . After 9escribed . Plates 6 parasite inocula and three wells of 107 parasite inocula. A final concentration of 120 nM IMP-1002 (3.4 x the 35 nM EC50) was added to a predominantly ring stage culture. The culture was fed daily with drug-containing complete medium for six days and every other day thereafter. The parasitemia was monitored and kept below 10 % over the next days to prevent overgrowth. Fresh RBCs  were added at day 7. At day 14, and weekly thereafter, the cultures were passaged (¾ cultured cells were replaced ¼ with fresh RBC and new medium) to prevent lysis. Drug pressure was maintained for 60 days.Prior to selection, genomic DNA was harvested and a frozen stock of the parental Dd2 parasite line was generated. Selection was initiated with three wells of 10nmt gene in P. falciparum 3D7 and Dd2 is identical (www.PlasmoDB.org) and was used for primer design. Following PCR amplification, the product was sequenced and cloned into pGEM-T Easy vector after ligation of a 3'-A overhang using Amplitaq polymerase to improve the sequence quality. Sequence data were analyzed using DNASTAR SeqMAN Pro and a consensus sequence for each of the parasite lines was fed into CLC Sequence Viewer 7 to create a nucleotide alignment. The nucleotide sequence was also translated into protein, which was aligned to the WT sequence using CLC Sequence Viewer 7.The genomic DNA sequence of the protospacer.com and cloned into the pDC2-cam-Cas9-U6-hDHFRyFCU-plasmid . Three biological replicates were generated in three different blood dilutions with three technical replicates each. WT parasitemia was determined following Giemsa staining after each cycle and used to determine the dilution factor to dilute all three lines down to 0.8%. Parasitemia was measured after each cycle using flow cytometry and Hoechst-stained parasites. Multiple t-tests were performed for each generation .4, 1 mM TCEP, 100 μM TBTA), the reaction was quenched with EDTA, and then precipitated proteins were washed with ice cold methanol as described previously  of each sample after gating out the RBC population using an RBC only sample as a control. The MFI of each sample was divided by the MFI of a control sample of synchronized rings with a known MFI of one nucleus.E. coli competent cells, using ampicillin for pET11a selection and chloramphenicol for the pRARE2 plasmid present in the Rosetta-gami DE3 cells to supply the tRNAs for rare codons used to codon-optimize PvNMT. PvNMT[WT] and PvNMT[G386E] were expressed following inoculation of a single colony overnight at 37°C in 5 mL Lysogeny Broth. The PvNMT[WT] protein was expressed in 8 x 500 mL auto-induction medium   and the PvNMT[G386E] protein in 19 L due to low yield. Auto-induction medium was combined with appropriate antibiotics and incubated with 1:100 dilution factor from the overnight culture at 37 C for 4 h. Temperature was then reduced to 18°C for 18-22 h. Bacterial cells were harvested by centrifugation and pellets from 4 L (for the WT protein) re-suspended in 180 mL of lysis buffer . For the PvNMT[G386E] protein cells from the 15 L were resuspended in 2x 300 mL, sonicated separately and then combined prior to application to the column. They were lysed using a BRANSON 550 sonication probe . The lysate was cleared by centrifugation at 48,384g RCF for 60 min at 4°C in a 25.50 JA rotor.The purification method has been adapted from earlier work . A regioFor large scale preparations the lysate was filtered through a 0.45 μM syringe filter and applied to a HisTRAP FF 5 mL column at 4°C and eluted with a linear gradient over 15 column volumes with 20 mM HEPES, 0.3 M NaCl, 5 % glycerol, 0.5 M imidazole; pH 7.0. Fractions containing the target protein were pooled and concentrated to about 2-3 mL using 10 kDa-cut off Pall centrifuge concentrators.2). The bound protein was eluted from the beads using 3x bead volume of elution buffer through gravity . Eluted proteins were analyzed using instant blue (Expedeon Prod # ISB1L). Nickel elution was filtered and concentrated using Centricon Plus 70 with a 30kDa cut-off.For small scale preps the lysate was incubated in batch with 20 mL of Ni-NTA Agarose beads (QIAGEN) equilibrated with lysis buffer in a 500 mL Duran bottle at 4°C for 1 hour. The beads were washed using a re-usable glass Fritted Funnel column and a pressure pump with 20x bead volume of wash buffer using a pressure pump  and applied directly to a HiTRAP Q HP 5 mL column  at 2 mL/min. Unbound sample was washed away with 5-8 column volumes (CV) at 2 mL/min for the 1 mL column and 3 mL/min for the 5mL column and eluted with 2 %-50 % of the elution buffer over 15 CV at 0.5 mL/min for the 1 mL column and 1 mL/min for the 5mL column. The 2 mL fraction (5 mL column) or 0.5 mL fractions (1 mL column) were analysed by applying 10 μL to an SDS PAGE gel and visualized using instant blue. For protein for crystallography, the His tag was cleaved from the PvNMT[WT] and PvNMT[G386E] proteins via 3C protease (His-MBP-3C) at a 1:50 protease: protein ratio overnight at 4°C in dialysis buffer  glycerol, 1 mM TCEP, and 0.025 % sodium azide). NMT was recovered by applying the dialysate to a second IMAC column. Fractions containing target protein were pooled and concentrated to 70 μL for the small scale or 4 mL for large scale and loaded onto Superdex 75 10/300 or 26/600 columns and eluted with size exclusion buffer  glycerol, 1 mM TCEP; pH 7.0). Fractions were analyzed on an SDS-PAGE gel and fractions containing the target protein were either pooled in the case of PvNMT[WT] or because of limited material kept separate for the PvNMT[G386E] protein. Fractions were concentrated and flash frozen and stored at – 80°C until further use.For small scale expression the purified protein was detected by Western blotting, using the anti-PvNMT antibody . For Western blots, proteins were transferred from the gel to nitrocellulose membrane with the iBlot™ Transfer System. Following blocking overnight at 4°C, membranes were incubated with primary antibody for 1 h at room temperature in blocking solution and incubated with secondary antibody  for 1 h in blocking solution. Detection was carried out using GE Healthcare ECL Western Blotting Detection reagents or BioRad Clarity Western ECL Substrate and visualized on a ChemiDoc MP BIO-RAD Imaging System.To measure the activity of the purified PvNMT[WT] and PvNMT[G386E], an assay was used with thiol-selective fluorescent dye (CPM) to monitor the formation of coenzyme A. The assay was performed as published in . Reagentmin + (ymax-ymin)/ (1+exp ((Tm-T)/slope)). Results were expressed as ΔTm in °C, where ΔTm is the difference between Tm in the presence of compound subtracted from that in the reference without compound present.Purified recombinant PvNMT[WT] and PvNMT[G386E] were diluted to 0.1 mg/mL in PBS buffer containing 10% glycerol and 1:1000 dilution of Sypro Orange in the presence or absence of 4 μM myristoylCoA. Test compounds were diluted to 500 μM in DMSO, and 0.5 μL of diluted compound was added to 49 ul of enzyme in a 96-well plate resulting in two technical replicates with a final volume of 20 μL and 1%  DMSO with a compound concentration of 5 μM. Each compound was tested twice to give two experimental replicates, each with two technical replicates. Reference thermal melting temperatures (Tms) were obtained using 1% DMSO without any compound present. The plates were subjected to a temperature gradient from 300K to 348K at a rate of 1K/min using a FluoDia fluorescent plate reader. Fluorescence data were acquired with an excitation filter of 486nm and an emission filter at 610nm, and the raw data were exported to Prism or Excel for analysis. Data were processed to identify the fluorescence maxima and minima, and the midpoints of melting curves were determined by fitting to a Boltzmann equation, where Tm is the temperature corresponding to the half maximum response.  equipped with research-grade CM5 sensor chips. PvNMT  was diluted to 0.0514 mg/mL in 10 mM sodium acetate pH 6.0. PvNMT[G386E]  was diluted to 0.0428 mg/mL in 10 mM sodium acetate pH 6.0. Each ligand was immobilized onto separate flow cells using amine-coupling chemistry. The surfaces of all four flow cells were activated for 7 min with a 1:1 mixture of 0.1 M NHS (N-hydroxysuccinimide) and 0.1 M EDC  propyl-N-ethylcarbodiimide) at a flow rate of 10 μL/min. PvNMT was immobilized at a density of 900 - 1100 RU onto flow cell 3, and PvNMT[G386E] at 600-1700 RU onto flow cell 4. Flow cell 1 was left blank to serve as a reference surface. All four surfaces were blocked with a 7 min injection of 1 M ethanolamine, pH 8.0. IMP-0320 was used as a positive control in the assay as it has been extensively used by GSK for routine NMT SPR assays.The analytes  were serially diluted 1:3 in DMSO, and then 1-3 μL of each dilution was dissolved in an assay buffer of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005 % Tween 20, 4 μM myristoylCoA to a final DMSO concentration of 1 %. To collect binding affinity data, the analyte concentrations (0-0.4 μM except from DDD85646: 0-1 μM) were injected sequentially over all four flow cells at a flow rate of 30 μL/min and at a temperature of 25°C. The complexes formed were allowed to associate for 200-700 sec and then dissociate for 600-2400 sec, dependent on the analyte kinetics observed. The surfaces were washed with an injection of 50 % DMSO between each analyte injection. The data were analyzed within Biacore T200 Evaluation Software v2.0.D PvNMT[G386E] to that of PvNMT [WT] was calculated for each compound dose response on the same chip within an experiment.Equilibrium sensorgram values were exported and the offset set to zero before fitting to a global ‘One site – Specific binding’ nonlinear regression model  in GraphPvNMT[WT] and PvNMT[G386E] were crystallized using the sitting drop vapor diffusion method at 7.8 mg/mL and 12.2 mg/mL respectively against a focused screen generated from a published literature condition . Drops c4B14) in the same crystal form as a search model. The structures were refined in Phenix (X-ray diffraction data was collected at the LS-CAT beamline 21-ID-F at the Advanced Photon Source. Collection information and processing statistics are reported in n Phenix , with man Phenix . Qualityn Phenix . Figuresn Phenix  and CCP4n Phenix . Inhibitn Phenix A were ge50s EC50, Hill coefficients and confidence intervals, was done using four-parameter [Inhibitor] vs. response. For KD determination through SPR in Data were analyzed using Prism 7.0 by GraphPad. Quantitative data are presented as mean ± standard deviation. Dose response curve fitting in 6MAY, 6MAZ, 6MB0, and 6MB1.The PDB files that support the findings of this study have been deposited in Protein Data Bank under accession codes


Dimocarpus longan Lour.) pericarp, the main by-product of aril and pulp processing, is abundant in phenolic compounds and worthy of further utilization. The present work firstly reported HPLC analysis and in vitro antioxidant evaluation of longan (cv. Shixia) pericarp-derived phenolics (LPPs), the purified longan pericarp extract (LPE), as well as their cytotoxic effect on lung cancer cell line, A549. The results indicated that the purified LPE had significant amounts of phenolics, with content of 57.8 ± 0.6 mg of gallic acid equivalents per gram of dry longan pericarp (mg GAE·g−1 DLP), which consisted of six phenolic compounds (A–F), including protocatechuic acid (A), isoscopoletin (B), quercetin (C), ellagic acid (D), corilagin (E), and proanthocyanidins C1 (F). Antioxidant assays showed that LPPs (10 μM) and LPE (1.0 mg·mL−1) had certain antioxidant activities, in which corilagin (E) possessed the best DPPH radical scavenging rate 71.8 ± 0.5% and •OH inhibition rate 75.9 ± 0.3%, and protocatechuic acid (A) exhibited the strongest Fe2+ chelating ability 36.4 ± 0.7%. In vitro cytotoxic tests suggested that LPPs had different effect on A549 cell line, in which corilagin (E) exhibited potent cytotoxicity with an IC50 value of 28.8 ± 1.2 μM. These findings were further confirmed by cell staining experiments.Longan ( Dimocarpus longan Lour.), one of the most popular fruits in temperate and sub-tropical regions, has a succulent and edible aril, with delicious flavor and health effects, as well as pharmacological properties, such as treating or relieving insomnia, neural pain, swelling, hyperglycaemia, improving women’s health after giving birth to a child and increasing the immunomodulatory capacity -6-phenyl-phenanthridinium diiodide) staining . The strAll NMR experiments were run on a Bruker Avance 600 MHz spectrometer equipped with a 5-mm triple resonance (HCN) cold probe. Positive HR-ESI-MS spectra were record on a Bruker micrOTOF-Q II mass spectrometer. Acetonitrile, formic acid and water used in HPLC system were of chromatographic grade and other chemicals were analytical.g  and retained on a normal atmosphere column with 500 mL macro-porous absorbent resin D101 , followed by eluting with three bed volumes of 80% alcohol. The collected elution was concentrated under reduced pressure and lyophilised. The afforded phenol-enriched extract was dissolved in methanol, with a final concentration of 10 mg·mL−1 and preserved at 4 °C before HPLC analysis.Mature longan (D. longan Lour. cv. Shixia) was grown in Foshan (China) and purchased from fruit market in Hangzhou City (China). The pericarp was manually separated from the whole fruit and freeze-dried . Dry longan pericarp (DLP) was ground and then passed through a 40 mesh sieve. About 50 g of DLP powder was extracted, with 500 mL of 60% aqueous ethanol, using a microwave and ultrasonic-assisted extractor  for 20 min. The obtained LPE was concentrated under reduced pressure and lyophilized . After re-dissolving in 500 mL of 20% ethanol solution, the resulting extract was centrifuged for 20 min at 50,000× w/v) was added to samples with different concentrations in a 10-mL volumetric flask. After 5 min, 0.1 mL of Folin-Ciocalteu reagent  was added, and the volume was increased to 10 mL, with deionized H2O. After incubation at 30 °C for 1 h, the absorbance was recorded at 750 nm on a Hitachi-UV-3000 spectrometer , and compared to a gallic acid calibration curve. Triplicate tests were conducted for each sample and the total phenolic content was expressed as milligram of gallic acid equivalents per gram of DLP (mg GAE·g−1 DLP).The total phenolic content of purified LPE was determined, using the modified Folin–Ciocalteu approach ,17. Two −1 as following gradient procedure: 0–25 min, 10 to 25% B; 25–50 min, 25% B; 50–70 min, 25 to 30% B; 70–90 min, 30 to 100% B; 90–100 min, 100% B; 100–110 min, and 100–10% B. All solvents were filtered with a 0.45 µm membrane filter and each injection volume was 5 µL. Quantitative analysis of LPPs was carried out using the external standard method described before [−1 DLP.Six phenolic standards including protocatechuic acid (A), isoscopoletin (B), quercetin (C), ellagic acid (D), corilagin (E) and proanthocyanidins C1 (F), and the positive control 5-fluorouracil were purchased from Sigma-Aldrich Chemical  and respectively dissolved in methanol with the initial concentration of 1.0 mM. HPLC analysis of LPPs was performed using a HPLC system  equipped with a 717plus auto-sampler and a 2487 UV-visible detector on a HPLC column  . The detection wavelength was set at 280 nm. The temperature of column was maintained at 20 °C. A gradient solvent system consisting of solvent A (0.1% aqueous formic acid) and solvent B (acetonitrile) was used at 0.5 mL·mind before . Triplic−1) was added to 100 μL of 0.2 mM DPPH in a 96-well plate, then incubated in the dark room temperature (25 °C) for 30 min. The absorbance of the mixture was measured at 517 nm by enzyme-linked immunosorbent assay plate reader . DPPH radical scavenging activity was calculated as follows:IDPPH is the DPPH radical scavenging rate (%), A0 is the absorbance of a negative control, A1 is the absorbance of each sample. All measurements were performed in triplicate.To assess the radical scavenging capacity of LPPs and the purified LPE, DPPH radical scavenging activity was investigated according to the procedure reported by Villaño et al. ,24. 100 −1) was incubated with 1.0 mL phenanthroline (2.5 mM), 1.0 mL distilled water, 1.0 mL ferrous sulfate (2.5 mM) and 1.0 mL hydrogen peroxide (20 mM) in phosphate buffer  for 90 min at 37 °C. The absorbance was measured on a spectrophotometer  at 536 nm. The ability to scavenge hydroxyl radical was calculated using the following equation:A1 is the absorbance of reaction mixture of hydrogen peroxide (H2O2), A2 is the absorbance of reaction mixture of distilled water, and A3 is the absorbance of reaction mixture of each sample. All measurements were performed in triplicate.Hydroxyl radical (•OH) assay was performed according to the procedure reported by Sun et al. ,26. The −1) was mixed with 100 mL FeCl2  and 1.7 mL deionized water. Then, 200 μL of ferrozine (5 mM) was added into the mixture, and fully mixed. The solution was then incubated at 25 °C for 10 min. The absorbance was measured using a spectrometer  at 562 nm with ethylenediamine tetracetic acid disodium salt (EDTA-2Na) as a positive control. Ferrous ion chelating activity was calculated as follows:A0 is the absorbance of the control (deionized water instead of sample); A1 is the absorbance of the sample; A2 is the absorbance of the sample only (deionized water instead of ferrous chloride). All measurements were performed in triplicate.Ferrous ion chelating capability was measured with the method described by Wang et al. . A quant−1) penicillin-streptomycin  followed by incubation at 37 °C in a humidified 5% CO2 incubator . The inhibitory effect of LPPs and LPE on cell proliferation was performed according to our previous report [3 per well were treated for 24 h with chemicals at different doses  or the purified LPE at different concentrations  with 5-fluorouracil as the positive control. Each well was added with 10 μL of cell counting kit-8 (CCK-8) solution , and the cells were further incubated for 1 h. Then the medium containing CCK-8 was removed and 100 μL of DMSO was added to each well. The plate was gently shaken for 15 min to dissolve the formazan crystals  and the absorbance was measured at 450 nm on a ELISA . The percentage viability was calculated using the following formula:IC is the percentage viability, sampleOD is the absorbance of sample group (10 µL CCK-8 reagent + 10 µL sample solution + 90 µL cell suspension), negativeOD is the absorbance of (10 µL CCK-8 reagent + 10 µL cell medium + 90 µL cell suspension), blankOD is the absorbance of blank group (10 µL CCK-8 reagent + 10 µL sample solution + 90 µL cell medium). The concentration of LPPs and the purified LPE needed to inhibit cell growth by 50% (IC50) was calculated from the dose response curves for each cell line [Human NSCLC A549 cell line was obtained from Cancer Research Center of Hangzhou Normal University . Firstly, these cells were maintained in RPMI-1640  medium consists of 10% FBS  and 1%   and kept in dark at 37 °C for 30 min, after the incubation, washing with buffer, add 1 mL culture [The logarithmic phase of A549 cells were washed with phosphate buffered saline (PBS) and digested with trypsin. When reaching a density of 5 × 10 culture ,30. All p < 0.05 were accepted as significant.All values were given as means ± standard and analyzed by SPSS software . A value of 2+ chelating ability (36.4 ± 0.7%). In vitro cytotoxic evaluation indicated that corilagin (E) also exhibited the strongest inhibitory effect on the growth of A549 cells, with an IC50 value of 28.8 ± 1.2 μM. Fluorescence staining experiments also confirmed that compounds E and F possess potent anti-proliferative activity against A549 cell. These findings would improve comprehensive utilization of longan (cv. Shixia) pericarp to make value-added products, including antioxidant food and pharmaceutical agents.The present work firstly reported on HPLC analysis and antioxidant activities of the purified LPE and LPPs as well as their cytotoxic effect on A549 cell line. These results suggested that the purified LPE is rich in phenolic compounds including protocatechuic acid (A), isoscopoletin (B), quercetin (C), ellagic acid (D), corilagin (E) and proanthocyanidins C1 (F). The antioxidant assay suggested that the purified LPE had certain capacity of scavenging DPPH•, inhibiting OH radical, and chelating ferrous ion. Compound E was shown to be the best antioxidant agent with DPPH• scavenging rate (71.8 ± 0.5%) and OH• inhibition rates (75.9 ± 0.3%) at 10 μM. And compound A possessed the strongest Fe


The catalysts were characterized by X-ray diffraction (XRD), laser Raman, transition electron microscope (TEM), scanning electron microscope (SEM), energy dispersive X-ray (EDX), hydrogen temperature programmed reduction (H2-TPR), and Brunauer-Teller-Emmett-Teller (BET) surface area techniques. CuFe2O4 was active for pinene oxidation using tertiary butyl hydroperoxide (TBHP) to pinene oxide, verbenol, and verbenone aroma oxygenates. Under optimized reaction conditions, the spinel CuFe2O4 catalyst could afford 80% pinene conversion at a combined verbenol/verbenone selectivity of 76% within the reaction time of 20 h. The changes in catalyst synthesis solvent composition ratios induced significantly varying redox, phases, and textural structure features, which resulted in various catalytic enhancement effect. Characterization results showed the spinel CuFe2O4 catalyst possessing less than 5 wt% impurity phases, Cu(OH)2, and CuO to afford the best catalytic performance. The CuFe2O4 catalyst was recyclable to up to five reaction cycles without loss of its activity. The recyclability of the bimetal CuFe2O4 oxide catalyst was simply rendered by use of an external magnet to separate it from the liquid solution.This study report on the synthesis of spinel CuFe The chemistry of aroma, flavor, and fragrance for cosmetic, therapeutic, and food ingredients constitute the backbone of the fine and specialty chemical industry . These kSeveral catalytic systems based on noble metals  and transition metals  have been investigated for their catalytic activity in the oxidation of pinene to aroma oxygenates ,18,19,200, Cu+1, and Cu+2) to facilitate the free-radicals catalyzed oxidation reactions [3O4), hematite (α-Fe2O3), maghemite (γ-Fe2O3), and epsilon (ε-Fe2O3) as most common stable phases [2O4) has been studied extensively in organic synthesis [2O4 catalyst, we further explore its catalytic activity in the oxy-functionalization of allylic inert C–H such as pinene oxidation to aroma oxygenates. The spinel CuFe2O4 can crystallize into two different phases during synthesis; namely, cubic and tetragonal structures. These crystal structure symmetries depend on the cations of Cu2+ and Fe3+ distribution in the tetrahedral (A) and octahedral (B) sites of the spinel CuFe2O4 structure-framework [The use of copper (Cu) and iron (Fe) metals in heterogeneous catalysis is well reported for liquid-phase oxidation of hydrocarbons into alcohols, ketones, aldehydes, and carboxylic acids ,22,23,24eactions ,26. On teactions ,28. Irone phases ,30. The ynthesis ,33,34,35ynthesis ,37,38. Bramework . Also, t2O4 catalysts by the design approach using different compositions ratio of solvent (ethylene glycol and water) in the presence of surfactant agent. Such synthesis methodology allowed the control formation of spinel CuFe2O4 with different structure morphologies, textural, phases compositions, and redox properties. The prepared powder catalysts were evaluated for their catalytic activity in the oxidation of pinene to aroma oxygenates such as pinene epoxide, verbenol, and verbenone. The structure of the catalysts were elucidated by using X-ray diffraction (XRD), laser Raman spectroscopy (LRS), Brunauer–Emmett–Teller (BET), field emission scanning electron microscope (FESEM) coupled to energy dispersive X-ray (EDX), high-resolution transition electron microscope (HRTEM), and hydrogen-temperature programmed reduction (H2-TPR).In this study, we report on the synthesis of spinel CuFe3)2·3H2O, 99.8%), iron nitrate nonahydrate (Fe(NO3)2·9H2O, 99.8%); polyvinyl pyrrolidone , ammonium hydroxide , ethylene glycol ; commercial CuFe2O4; tertiary butyl hydroperoxide ; pinene (99.0%); verbenol (99.8%); verbenone (99.8%); pinene oxide (99.99%); nitrobenzene (99.8%). Merck Millipore  produced deionized water was used.The reagents used were of analytical grade purchased from Sigma Aldrich  and Minema . Copper nitrate trihydrate  was dissolved in a 100 mL (1EG:1H2O molar ratio) solvent mixture in a 250 mL round bottom flask and stirred until the color of the solution was clear at 100 °C under reflux. Then, 4.62 g of Cu(NO3)2·3H2O and 5.70 g of Fe(NO3)2·9H2O were added and the mixture was continuously stirred to ensure complete dissolution of the metal salt precursors. The pH of the reaction solution was adjusted to 8.0 by dropwise addition of 28% aq. NH3·H2O. The reaction mixture was then stirred continuously under reflux at 100 °C for 20 h to precipitate the catalyst slurry nanoparticles. Similar procedure was repeated for other solvent composition ratios of 1EG:4H2O and 4EG:1H2O·CuFe2O4 catalysts synthesis. The resulting catalysts solid slurries were recovered by evaporation, and oven dried at 150 °C for 12 h. The dried catalysts were calcined in a muffle furnace under air from 30 °C to 450 °C and held for 3 h at a heating rate of 5 °C/min. In addition, single CuO and Fe2O3 were synthesized following the same procedure. The final catalysts were denoted: CuFe-1 ; CuFe-2 (1EG:1H2O), CuFe-4 (4EG:1H2O), and CuFe-3 (1EG:4H2O), respectively.The synthesis of CuO, Fe2) sorption at 77 K, using a Micromeritics TRISTAR 3000  surface area analyzer. Prior to N2 physisorption experiments, the samples were degassed at 120 °C for 12 h under a continuous flow of N2 gas to remove surface-adsorbed contaminants. The H2-TPR analysis was studied on a Micromeritics Autochem II AC2920  instrument. Prior to the analyses of the samples, the surfaces of the catalysts were first cleaned by being subjected to flow of argon (Ar) at 100 °C for 1 h. Then, the reduction of the catalysts was carried out under the flow of 10 vol.% of H2 in Ar at a heating rate of 10 °C/min from 50 °C to 800 °C and total gases flow rate of 50 mL/min. The H2 consumption was monitored by a calibrated thermal conductivity detector (TCD).Powder XRD measurements were recorded on a PAnalytical XPERT-PRO diffractometer measurement, using Ni filtered CuKα radiation (λ = 1.5406 Å) at 40 kV/50 mA. The diffraction measurements were collected at room temperature in a Bragg–Brentano geometry with scan range, 2θ = 10°–90° using continuous scanning at a rate of 0.02°/s. The morphology and particles dimensions were measured by HRTEM  operated at an accelerating voltage of 200 kV. Before HRTEM analysis, the samples were prepared by suspending the solid in ethanol under ultra-sonication for 30 min. Afterward a drop was extracted and placed on carbon-coated copper grid for TEM measurements. The microstructure morphologies and elemental analysis were characterized by means of FESEM coupled with EDX, respectively. The samples were sputter coated with carbon to avoid charging before performing FESEM measurements. The BET surface area, pore size, and volume of the catalysts were measured by nitrogen  flask equipped with magnetic stirrer and reflux condenser. To 50 mL RB, pinene , acetonitrile solvent (10 mL), and catalyst (0.10 g) were added together. The reaction temperature was controlled at 80 °C by immersing the reaction mixture solution inside hot oil bath. After the reaction solution temperature of 80 °C has been stabilized, 70% aq. TBHP oxidant  was then added and immediately the start of the reaction time was recorded. Samples were withdrawn accordingly from the reaction mixtures and analyzed using gas chromatograph (GC).The high-pressure catalytic oxidation experiments were performed in a 50 mL stainless-steel autoclave reactor  equipped with Teflon liner. To the reactor, pinene , 70% (aq.) TBHP , acetonitrile solvent (30 mL), and catalyst (0.30 g) were added together. The reaction temperature was controlled at 90 °C. Samples were withdrawn accordingly from the reaction mixtures and analyzed using GC.The composition of pinene oxidation products mixture was analyzed by Agilent GC  equipped with a flame ionization detector (FID) and a capillary column . The injection temperature was 200 °C and FID 300 °C. The separation column oven temperature was from 70 °C to 200 °C at a heating ramp rate of 10 °C/min, followed by the isothermal hold at 200 °C for 5 min. Nitrobenzene was used as the internal standard, while authentic standards such as pinene oxide, verbenol, and verbenone were used for identification of the targeted peaks.2O4 catalysts synthesized using different EG:H2O solvent compositions, including the commercial (CuFe-1), single Fe2O3, and CuO catalysts. The formation of CuO was confirmed to be a monoclinic phase (JCPDS48-1548). The XRD peaks at 2θ = 18.6°; 30.4°; 33.5°; 35.7°; 37.4°; 43.5°; 49.9°; 53.7°; 54.5°; 57.2°; 62.7°; 74.3° were indexed to those of magnetite Fe2O3 with tetragonal and rhombohedral crystal structure. The XRD of spinel CuFe2O4 is usually characterized by either or both the cubic (space group Fd3m) and distorted tetragonal (space group I41) crystal symmetry structure patterns [2O4 crystal symmetry structures are distinguished by typical XRD peaks at 2θ = 30.2°; 35.6°; 62.8° for the cubic, while tetragonal is 2θ = 34.7°; 35.9°; 62.2°. The CuFe-1 catalyst showed the XRD peaks at 2θ = 18.6°; 30.3°; 35.6°; 36.4°; 43.5°; 57.3°; 62.9°; 74.9°, which correspond to miller indices of (002); (200); (211); (202); (220); (303); (224); (413), respectively. Moreover, the XRD patterns of CuFe-1 were relatively similar to those of synthesized CuFe-2, CuFe-3, and CuFe-4 spinel catalysts. Noticeably, the synthesized CuFe2O4 catalysts showed varying XRD peaks intensities, which were induced by the different solvent composition ratios effect on NPs nucleation growth rates. In addition, the influence of synthesis solvent compositions on XRD crystallographic structure parameters of CuFe2O4 catalysts such as phases compositions, lattice planes, crystallite size, diffraction spacing, and crystallite strain were evaluated using the Rietveld refinement analysis of the obtained XRD data 2 phase in addition to spinel CuFe2O4 phase , Eg, F2g (2), F2g (3), and A1g, respectively [−1 corresponding to Ag, Bg (1), and Bg (2), respectively [2O3 were ascribed to tetragonal crystal symmetry structure, while CuO was monoclinic phase, thus complimenting the XRD results [2O to EG of (1:1) and (4:1), respectively, they displayed five highly intense Raman vibration active modes at 225, 280, 400, 480, 615, and 690 cm−1. These vibrational active modes were predominantly similar to those obtained for Fe2O3. This indicated that the Fe2O3 had more Raman vibrational modes active than CuO. As a result, the intensity of the Fe2O3 phase was predominant in the CuFe2O4 catalysts. On the other hand, for CuFe-4 catalyst with ratio of H2O to EG (1:4), its Raman active modes were significantly weak in intensity. This was attributed to the observed XRD structural evolution, which was shown to lead to the destruction of the tetragonal and cubic crystal symmetry structure of the spinel CuFe2O4 phase. This structure deviation of CuFe-4 could be associated with its observed structural disorder due to the formation of 4.3% Cu(OH)2 phase in addition to spinel CuFe2O4 phase as confirmed by the XRD crystallographic Rietveld refinement results  and (004), respectively, whereas r, 5.891 and 6.681 nm−1 were not included in 2θ range  was used. As can be seen in in 2θ range d. The HR2θ range .2/g and pore volume of 0.022 cm3/g. The low SA of CuO was due to the observed particles agglomeration into large size. Inversely, Fe2O3 afforded the highest SA of 48.66 m2/g and better pore volume of 0.156 cm3/g than CuO did. The commercial CuFe-1 had the SA of 32.38 m2/g and 0.065 cm3/g pore volume. The synthesized CuFe2O4 catalysts by varying EG:H2O solvent composition ratio exhibited a profound influence on both SA and pore volume, which were better than the CuFe-1 catalyst  and porosity results of the catalysts are summarized in catalyst . CuO shoensation . Further results .2O4 catalysts. The TPR of CuO showed a single reduction of Cu+2→Cu0 in the temperature range of 100–200 °C. On the other hand, the TPR profile of Fe2O3 displayed two-stage reduction peaks in the temperature ranges of 125–280 °C and 300–500 °C, respectively. These different reduction temperatures showed Fe2O3 to undergo multi-step reduction that can be associated with Fe+3→Fe+2 and Fe+2→Fe+1/Fe0. Moreover, Fe2O3 reduction peaks were broad and stretched over a wide temperature range, which could be an indication of NPs of varying sizes. The TPR of commercial CuFe-1 displayed two distinct reduction peaks at 125–160 °C and 220–350 °C which are attributed to the octahedral and tetrahedral M2+ cations sites reductions, respectively. Furthermore, the use of different solvent compositions to synthesize CuFe2O4 catalysts was shown to result in various structure interaction strength of the two metal oxides as demonstrated by their shift in peaks reduction temperature patterns in a range of 110–505 °C  < CuFe-2 (110 °C) = CuFe-4 (110 °C) < CuFe-3 (120 °C) < CuFe-1 (125 °C) < Fe2O3 (130 °C) maximum peak points. These redox reactivity patterns of CuFe2O4 catalysts were induced by variations in synthesis solvent compositions; thus, different structure formations could bring about probable significant differences in catalytic enhancement effect.0–505 °C . The TPR2O4 catalysts were evaluated in pinene oxidation reaction to various aroma oxygenates, including pinene oxide, verbenol, and verbenone. Firstly, the blank test reaction of pinene and TBHP oxidant in acetonitrile solvent was carried out at 80 °C under atmospheric conditions. A pinene conversion of 3.3% was achieved for non-catalytic reaction at respective selectivities of 55.0% (pinene oxide), 9.1% (verbenol), and 6.3% (verbenone)  and 10.3% (Fe2O3). CuO showed respective selectivities of 60.3% (pinene oxide), 12.3% (verbenol), and 8.2% (verbenone), while for Fe2O3, they were 23.4% (pinene oxide), 17.3% (verbenol), and 38.4% (verbenone) (2O3 compared to CuO. The improved selectivity to ketone (verbenone) for Fe2O3 was ascribed to the presence of prominent Lewis acidic sites associated with iron oxide catalysts, which could facilitate further oxidation of verbenol to verbenone in similar manner reported for alcohols oxidation to ketones [2O4 catalysts, they all showed better pinene conversion amounts than individual Fe2O3 and CuO catalysts  induced by varying catalyst synthesis solvent compositions. The highest pinene conversion amount was obtained with CuFe-4 catalyst, which also gave better combined verbenol/verbenone selectivity ; CuFe-2 (98.2%); CuFe-3 (98.2%) and CuFe-4 (94%). Additionally, it must be highlighted that the CuFe-4 catalyst also showed the formation of 4.3% of Cu(OH)2 and 1.7% CuO phases compositions in addition to the spinel CuFe2O4 phase. It must also be borne in mind that the metal-hydroxyl coordinated complex structure, M-(OH)2  is the indication of the presence of some Brønsted basic sites, which contains the active hydroxide ion (OH−). Accordingly, the OH− functional group is capable of accepting the proton . Thus, the OH− could perhaps be exerting some unique catalytic active sites influence for the CuFe-4 catalyst arising from the additional contribution of the Cu(OH)2 phase in conjunction to spinel CuFe2O4 phase. Arguably, this could somehow be influential in the reactivity of abstracting the protonic H+ from R-H organic substrate  in concomitance to coupled Fe2+/Fe3+ and Cu+/Cu2+ redox effect, contributing to initiate the chain free-radicals activation of R-H to R and H+ with enhanced oxidation activity and rates compared to other catalysts. Although this proposition is a postulation, the probability of it occurring could be associated with the high catalytic activity of CuFe-4 catalyst, which is composed of Cu(OH)2 compared to other catalysts. Studies are under progress to follow up on the controlled formation of Cu(OH)2 structure phase in the spinel CuFe2O4 catalysts, characterization, and detailed probing of its actual catalytic effect with respect to pinene oxidation mechanism and products selectivity distribution. Furthermore, all synthesized CuFe2O4 catalysts were characterized by the presence of segregated phase of CuO of up to 1.8% content, while commercial CuFe-1 did not possess. As a result, the enhanced catalytic performance observed for CuFe-4 catalyst is associated with additional catalytic contribution from impurity phases of Cu(OH)2 and CuO. This invoked the probable catalytic activity enhancing of spinel CuFe2O4 catalyst by the creation of additional surface quantitative impurity amounts in the form of other oxide phases or metal hydroxyl groups. In fact, the presence of Cu(OH)2 could be facilitating the high oxygen mobility and redox activity of CuF-4 better than the other CuFe2O4 catalysts that showed no formation of Cu(OH)2 phase. This phenomenon could be correlated to the surface oxygen reactivity of CuFe2O4 catalysts observed in the H2-TPR results (2O) compositions. Based on the amount of the major products obtained, the tentative reaction route mechanism to verbenol and verbenone formation can be illustrated with the plausible reaction The catalytic activities of CuFerbenone) , entry 1ectivity , the res results . AccordiN,N-dimethylformide (DMF). Following the activity screening of the most active catalyst sample, the CuFe-4 was selected as the best to further evaluate its catalytic performance under the effect of various reaction parameters including, (i) solvents, (ii) temperature, (iii) catalyst recyclability, and (iv) comparison of pinene oxidation reaction under different reactor conditions. The reaction solvent is one of the important reaction constituents that can facilitate efficient mixing of reactants by improving their solubility and reactivity interactions. Moreover, the solvent can have a profound influence on conversion performance of the catalyst, including products selectivity by enhancing on catalyst surface adsorption/desorption mass transfer of the reactants. Hence, the catalytic performance of CuFe-4 in the pinene oxidation reaction was evaluated under various solvents, including acetonitrile, water, ethanol, tetrahydrofuran (THF), and Temperature has a profound effect on chemical reaction rates; thus, it could impact substrate conversion and products distribution spectrum. For this reason, the effect of reaction temperature on pinene oxidation rates was studied in a range of 60–100 °C. In The durability and stability of the solid heterogeneous catalyst under liquid-phase reaction medium is of prime importance in determining its potential industrial application. As a result, the recyclability performance of CuFe-4 catalyst was investigated in the oxidation of pinene under the same reaction conditions for five re-use reaction cycles. After each reaction recycle, the catalyst was washed using the acetonitrile solvent and air-dried in an oven at 100 °C for 5 h before use in the next reaction cycles. The results obtained for catalytic recyclability performance of CuFe-4 catalyst are illustrated in 2O2; thus, its utilization under pressurized condition will require suitable reactor configuration such as stainless steel. Moreover, the excellent performance of TBHP oxidant utilization under self-pressurized conditions has also been demonstrated by Gawade et al. [The choice of a reactor is a critical factor in the rational development of an efficient catalytic chemical conversion process in addition to the designed active catalyst. It can influence factors such as mass transfer , temperature distribution, and pressure for reactants solubility. For these reasons, the catalytic performance of CuFe-4 catalyst was evaluated in the pinene oxidation reaction under different reactor conditions, including (i) atmospheric batch glass flask, and (ii) high-pressure autoclave. e et al.  in the s2O4 catalysts with varying structure morphology, phases compositions, textural, and redox properties achieved by changing the synthesis solvent mixture (ethylene glycol:water) compositions. The CuFe2O4 was active for oxidation reaction of pinene to pinene oxide, verbenol, and verbenone aroma oxygenates using tertiary butyl hydroperoxide as oxidant. The activity of CuFe2O4 was profoundly influenced by the redox, phase compositions, and textural structure. According to the characterization results, the most active spinel CuFe2O4 catalyst possessed quantitative amounts of impurity phases, including Cu(OH)2 and CuO phases amounting to less than 5 wt% concentration, which were advantageous for high catalytic performance catalyst. Furthermore, these impurity phases enhanced better surface oxygen reactivity of CuFe2O4 catalyst based on redox TPR results, which could facilitate efficient high oxygen mobility during the liquid-phase oxidation reaction.In summary, we have described the synthesis of spinel CuFe


Curcuma longa and is known for its use in Chinese and Ayurvedic medicine. Turmeric is rich in curcuminoids, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Curcuminoids have potent wound healing, anti-inflammatory, and anti-carcinogenic activities. While curcuminoids have been studied for many years, not much is known about their effects on steroid metabolism. Since many anti-cancer drugs target enzymes from the steroidogenic pathway, we tested the effect of curcuminoids on cytochrome P450 CYP17A1, CYP21A2, and CYP19A1 enzyme activities. When using 10 µg/mL of curcuminoids, both the 17α-hydroxylase as well as 17,20 lyase activities of CYP17A1 were reduced significantly. On the other hand, only a mild reduction in CYP21A2 activity was observed. Furthermore, CYP19A1 activity was also reduced up to ~20% of control when using 1–100 µg/mL of curcuminoids in a dose-dependent manner. Molecular docking studies confirmed that curcumin could dock onto the active sites of CYP17A1, CYP19A1, as well as CYP21A2. In CYP17A1 and CYP19A1, curcumin docked within 2.5 Å of central heme while in CYP21A2 the distance from heme was 3.4 Å, which is still in the same range or lower than distances of bound steroid substrates. These studies suggest that curcuminoids may cause inhibition of steroid metabolism, especially at higher dosages. Also, the recent popularity of turmeric powder as a dilatory supplement needs further evaluation for the effect of curcuminoids on steroid metabolism. The molecular structure of curcuminoids could be modified to generate better lead compounds with inhibitory effects on CYP17A1 and CYP19A1 for potential drugs against prostate cancer and breast cancer.Turmeric, a popular ingredient in the cuisine of many Asian countries, comes from the roots of the  Curcuma longa, which belongs to the ginger family Zingiberaceae. Curcumin  is the most abundant of the curcuminoids and enhances wound healing, modulates angiogenesis and the immune system, and has anti-inflammatory, anti-oxidant, anti-infective and anti-cancer activities -pregnenolone, [7(N)-3H]-pregnenolone (12.6 Ci/mmol), and [3H]-androstenedione: Andros-4-ene-3, 17-dione, [1β-3H(N)]-, 250 µCi (9.25 MBq) were purchased from PerkinElmer . [3H]-17α-OH progesterone  (60-120 Ci/mmol 2.22-4.44 TBq/mmol) was obtained from American Radiolabeled Chemicals Inc. . Silica gel-coated aluminum backed TLC plates were purchased from Macherey-Nagel . The tritium-screens used for the autoradiography were purchased from Fujifilm . Turmeric extract capsules (from Curcuma longa) were obtained from the Finest Natural . Trilostane was extracted in absolute ethanol (EtOH) from tablets commercially available as Modrenal® . Abiraterone was purchased from Selleckchem . Anastrozole was obtained from AstraZeneca .Radiolabeled -pregnenolone was added to each well. For the determination of CYP21A2 activity, [3H]-17α-OH progesterone  was used as a substrate. After incubations with radiolabeled substrates, medium from each well was collected, and steroids were extracted in ethyl acetate:isooctane (1:1). Extracted steroids were concentrated under nitrogen and dissolved in 20 µL of trichloromethane for separation by thin-layer chromatography on silica gel thin layer chromatography (TLC) plates . Radiolabeled steroids were quantified by autoradiography on a Fuji FLA-7000 PhosphorImager . Quantification of steroids was done using MultiGauge software .Human adrenal NCI-H295R cells were seeded in 6-well tissue culture plates at a density of 1 × 10rotocols ,56,89,90g for 5 min to pellet the cells. Afterward, the cell pellet was suspended in 100 mM Na3PO4 (pH 7.4) containing 150 mM KCl, and the cells were lysed by sonication. Unbroken cells and mitochondria were pelleted by centrifugation at 14,000× g for 15 min at 4 °C. Microsomes containing endoplasmic reticulum were collected by ultracentrifugation at 100,000× g for 90 min at 4 °C and resuspended in 50 mM K3PO4 (pH 7.4) containing 20% glycerol [JEG3 cells were collected near confluency and washed with cold PBS. The cell suspension was then centrifuged at 1500× glycerol . The pro3H]-androstenedione as a radioactive tracer. The aromatase reaction was initiated by the addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to 1 mM final concentration and incubations were done at 37 °C with constant shaking for 1 h. Afterward, 800 µL of a solution containing 5% charcoal with 0.5% dextran was added to each reaction mixture and mixed by vortexing followed by centrifugation at 15,000× g for 10 min. From each reaction, 500 µL aliquots were taken for the measurement of radioactivity by scintillation counting using the Rotiszint Universal Cocktail , and aromatase activity was calculated as described previously [The aromatase activity of CYP19A1 was measured by calculating the release of tritiated water from the substrate during aromatization. Assays of aromatase activity were carried out using 40 µg of microsomes from JEG3 cells in 100 mM potassium phosphate buffer (pH 7.4) containing 100 mM NaCl in a final volume of 200 µL. Different concentrations of curcuminoids were added to reaction mixtures, and ethanol was used as the control. A known inhibitor of CYP19A1, anastrozole was included in some reactions as a positive control. Assays were performed using 50 nM androstenedione as a substrate and contained ~20,000 cpm of [eviously ,94.The published 3D structures of human CYP17A1, CYP19A1, and CYP21A2 were obtained from the protein data bank (PDB) database. We made in-silico calculations and structure analysis with YASARA . For docp < 0.05 and ** p < 0.001.For the statistical analysis, Microsoft Excel and GraphPad Prism  were used. Data are presented as the mean and standard deviation of 3 independent replicates. One-way ANOVA was used to calculate the differences between the samples with the corresponding controls. Significance cutoffs for experiments was set at * Computational docking from our experiments showed that curcumin could bind into the active sites of steroid metabolizing P450s. The inhibition of CYP17A1 and CYP19A1 by curcuminoids provides a template for structure modification to produce effective and safe compounds that can target prostate cancer as well as breast cancer. Curcumin-based compounds can be further optimized for delivery as nano formulations, which have been tried in laboratory animals ,100,101


Estrogens have been shown to rapidly regulate local signalling at synapses and within the nucleus. The result of these signalling events is to rapidly modulate synapse structure and function, as well as epigenetic mechanisms including histone modifications. Ultimately these mechanisms are thought to contribute to long-lasting changes in neural circuitry, and thus influence cognitive functions such as learning and memory. However, the mechanisms by which estrogen-mediated local synaptic and nuclear signalling events are coordinated are not well understood. In this study we have found that the scaffold protein afadin, , undergoes a bi-directional trafficking to both synaptic and nuclear compartment in response to acute 17β-estradiol (estradiol) treatment, in mixed sex neuronal cultures derived from fetal cortex. Interestingly, nuclear accumulation of afadin was coincidental with an increase in the phosphorylation of histone H3 at serine 10 (H3S10p). This epigenetic modification is associated with the remodeling of chromatin into an open euchromatin state, allowing for transcriptional activation and related learning and memory processes. Critically, the cyto-nuclear trafficking of afadin was required for estradiol-dependent H3S10p. We further determined that nuclear accumulation of afadin is sufficient to induce phosphorylation of the mitogentic kinases ERK1/2 (pERK1/2) within the nucleus. Moreover, nuclear pERK1/2 was required for estradiol-dependent H3S10p. Taken together, we propose a model whereby estradiol induces the bi-directional trafficking of afadin to synaptic and nuclear sub-compartments. Within the nucleus, afadin is required for increased pERK1/2 which in turn is required for H3S10p. Therefore this represents a mechanism through which estrogens may be able to coordinate both synaptic and nucleosomal events within the same neuronal population. •17β-estradiol targets afadin to membrane and nuclear subcompartments.•Histone H3 is rapidly phosphorylated by 17β-estradiol.•Histone H3 phosphorylation by 17β-estradiol requires afadin nuclear accumulation.•17β-estradiol-mediated ERK1/2 activation is required for histone H3 phosphorylation. The rapThere is accumulating evidence that post-translational modifications of histone proteins is a critical mechanisms for the remodeling of chromatin . The phoRecently it have become apparent that proteins residing at synaptic or cytoplasmic locations can translocate to the nucleus in response to specific stimuli . Protein22.1The following antibodies were purchased: GFP mouse monoclonal (MAB3580: 1:1000), NeuN mouse monoclonal , phospho-histone H3 serine 10 mouse (H3S10p) monoclonal  and myc rabbit polyclonal (06–549: 1:500) were from Millipore; phospho-p44/42 MAPK  rabbit monoclonal , ERK1/2 mouse monoclonal  were from Cell Signalling Technologies; GFP chicken polyclonal  and histone 3  rabbit polyclonal (ab1791: 1:2000) were from Abcam; myc mouse monoclonal ; L/S-afadin rabbit polyclonal , β-actin mouse monoclonal  were from Sigma; DAPI was from Life Technologies. The specificity of the L/S afadin antibody has been previously demonstrated by shRNA knockdown and in two independent conditional knockout animals . Estradi2.2d-lysine , at a density of 3 × 105/well equal to 857/mm2. Neurons were cultured in feeding media: neurobasal medium (21103049) supplemented with 2% B27 (17504044), 0.5 mM glutamine (25030024) and 1% penicillin:streptomycin (15070063) . It should be noted that the neurobasal media contains phenol red, a compound with known estrogenic activity. Neuron cultures were maintained in presence of 200 μM d,l-amino-phosphonovalerate  beginning on DIV (days in vitro) 4 in order to maintain neuronal health for long-term culturing and to reduce cell death due to excessive Ca2+ cytotoxicity via over-active NMDA receptors : (in mM) 125 NaCl, 2.5 KCL, 26.2 NaHCO3, 1 NaH2PO4, 11 glucose, 5 Hepes, 2.5 CaCl2, 1.25 MgCl2, and 0.2 APV). ACSF was used to ensured that there were no interactions between the pharmacological compounds being used and unknown elements within the culture media that may indirectly affect the experimental outcome. For kinase inhibitor experiments, neurons were pre-treated for 30 min before application of E2 directly over neurons. All compounds were dissolved in either 100% ethanol or DMSO at a concentration of 10 or 1 mM, serially diluted to a 10X working concentration in aCSF, and applied directly to neuronal cultures. Final concentration of solvent was <0.01%: vehicle control was made up of solvent lacking compound diluted as test compounds. Treatments were allowed to proceed for indicated time before being lysed for Western blotting or fixed for immunocytochemistry (ICC).2.4Neurons were fixed in either 4% formaldehyde/4% sucrose PBS for 10 min, or in 4% formaldehyde/4% sucrose PBS followed by 10 min fix with Methanol pre-chilled to −20 °C. Coverslips were then permeabilised and blocked simultaneously in PBS containing 2% normal goat serum and 0.2% Triton-X-100 for 1 h at room temperature. Primary antibodies were added in PBS containing 2% normal goat serum for 2 h at room temperature, or overnight at 4 °C, followed by 3 × 10 min washes in PBS. Secondary antibodies were incubated for 1 h at room temp, also in 2% normal goat serum in PBS. Three further washes (15 min each) were performed before cells were incubated in DAPI if required. Finally, coverslips were mounted using ProLong antifade reagent (Life Technologies).2.5https://imagej.nih.gov/ij/)  as a z-series, or with a Zeiss Axio Imager Z1, equipped with an ApoTome using a 63× oil-immersion objective . Two-dimensional maximum projection reconstructions of images were generated and linear density calculated using ImageJ/Fiji (gov/ij/) . For mea2.6Synaptic and dendritic localization of afadin was quantified using MetaMorph . Images 2.73. Nuclear fractions were left on ice for 10 min before centrifugation at 6500 × g at 4 °C; resulting pellet was retained and washed in TEB, subject to centrifugation before being resuspended in 0.2 M HCl overnight at 4 °C. Samples were then centrifuged at 6500 × g at 4 °C and supernatant collected; HCl was neutralised with 2 M NaOH. Histone enriched nuclear fractions were then subjected to Western blotting; membranes were probed with appropriate histone antibodies.Subcellular fractions were prepared using the Proteo-Extract kit (EMD Biosciences) following the manufacturer's recommendations. Lysates were subjected to Western blotting; membranes were probed with the appropriate antibodies. Histone enriched nuclear fractions were prepared as follows. Treated neurons were collected in 150 μL ice cold homogenisation buffer: 320 mM sucrose; 5 mM Na4P2O7; 1 mM EDTA pH 8; and 10 mM HEPES pH 7.4 in the presence of protease and phosphatase inhibitors and homogenised by passage through a 25 gauge needle 12 times. The lysate was centrifuged at 800 × g for 10 min at 4 °C to yield the P1 (nuclear) fraction. P1 fractions were then resuspended in triton extraction buffer (TEB): PBS containing 0.5% Triton ×100, 2 mM AEBSF and 0.02% (w/v) NaN2.8All statistical analysis was performed in GraphPad. Differences in quantitative immunofluorescence, dendritic spine number were identified by Student's unpaired t-tests. For comparisons between multiple conditions, the main effects and simple effects were probed by one-way-ANOVAs followed by Tukey Post Hoc analysis. Error bars represent standard errors of the mean. Between 3 and 5 independent cultures were used for all experiments.33.1Previous studies have demonstrated that afadin is a dual residency protein present in both nuclear and cytosolic compartments, and specifically at synapses . Moreove3.2The rapid post-translational modification of H3 has been observed in response to synaptic activity as well as neuromodulators . In partNext we investigated whether H3S10p was associated with the cyto-nuclear shuttling of afadin, as the presence of this scaffold protein in the nucleus is required for activity-dependent phosphorylation of H3 at serine 10 . Therefo3.3Our data above suggest a relationship between afadin trafficking to the nucleus and H3S10p. This observation is consistent with the requirement of increased nuclear afadin for activity-dependent H3S10p . Therefo3.4One of the major kinases that mediates the rapid effects of estradiol is the mitogenic kinases, ERK1/2 . Interes3.5As estradiol rapidly increased pERK1/2 levels in the nucleus, we reasoned that this kinase may be part of the pathway resulting in increased H3S10p levels. Consistent with this, the mitogenic kinases MSK1 and RSK1, which directly phosphorylate H3 at serine 10, are direct targets of ERK1/2 . Therefo4In this study we have demonstrated that estradiol causes the bi-directional trafficking of afadin to synaptic and nuclear compartments within the same cell. Furthermore, we present evidence that estradiol phosphorylates histone H3 at serine 10, which is dependent on the nuclear accumulation of afadin. Interestingly, estradiol also caused an increase in pERK1/2 levels within the nucleus, which was mimicked by the overexpression of l-afadin. Moreover, we found that the MEK/ERK1/2 signalling cascade was required for estradiol-dependent increases in H3S10p levels. Taken together, we propose a model where afadin is bi-directional trafficked to synaptic/membrane regions and the nucleus in response to estradiol stimulation; in the nucleus, afadin is mediates estradiol-mediated increases in nuclear pERK1/2 and H3S10p  C. This The molecular mechanisms that enable neurons to translate signals generated at dendrites and synapses to the nucleus in order to regulate nucleosomal events are not well understood . One mecThe data we present in the study above is consistent with our previous work whereby following stimulation, afadin is trafficked to synaptic/membrane subcompartments, but also shuttles to the nucleus within the same cell . We haveMultiple studies have shown the that histone H3 is phosphorylated in response to a variety of stimuli. Importantly, this modification has been established to be sufficient to confer a change in chromatin from a condensed heterochromatin state to a euchromatin state; more amenable to gene transcription and associated transcriptional activation . H3S10p Previous studies have demonstrated that estrogens can regulate H3S10p to control cell proliferation in tumour and ovarian cells . HoweverA surprising finding in this study, is the potential link between afadin, ERK/2 and H3S10p. Previous studies have shown that estradiol rapidly increases pERK1/2 levels in the nucleus , and morTaken together, this study provides the first evidence that estradiol can phosphorylate histone h3 in neurons. More specifically, estradiol induces the cyto-nuclear trafficking of afadin and subsequent nuclear ERK1/2 activation of H3S10p under stimulated conditions. These data are consistent with a growing number of studies that demonstrate that estrogens are capable of modulating gene transcription and protein synthesis in a non-canonical manner . MoreoveK.J.S, I.A.W., R.E.G., P.R. and D.P.S. conducted and analysed experiments; D.P.S. designed and conceptualized the study; K.J.S, I.A.W. and D.P.S. contributed to the writing; D.P.S. oversaw the project.The authors report no conflict of interests.