Our results suggest that rodents show similarity to humans for studying the impact of CYP46A1 inhibitors and that rapid, local modulation of oxysterols can be achieved through CYP46A1 inhibition.The manipulation of cholesterol and its metabolites has been hypothesized to be therapeutically beneficial for mood disorders, neurodegenerative disorders, and epilepsies. A major regulator of cholesterol clearance and turnover in the central nervous system is CYP46A1, a brain enriched enzyme responsible for metabolism of cholesterol into 24S-hydroxycholesterol. Inhibition of this enzyme may negatively modulate NMDARs as 24S-hydroxycholesterol was shown to enhance NMDAR function. In addition, alterations of local cholesterol or other changes mediated by CYP46A1 activity could have important influences on central nervous system function. Here we demonstrate that humans and mice display brain region specific and similar CYP46A1 and 24S-hydroxycholesterol distribution. Treatment with distinct classes of CYP46A1 inhibitors led to central 24S-hydroxycholesterol reduction  CYP46A1). Although 24S-HC flux to the periphery is important for brain cholesterol turnover (The oxysterol 24S-hydroxycholesterol (24S-HC) is an enzymatically generated oxidation product of cholesterol. It is selectively synthesized in central nervous system (CNS) neurons by the cytochrome P450 enzyme cholesterol 24-hydroxylase (8-(oxazol-5-yl)-6-(trifluoromethyl)imidazopyridin-3-yl)methanone  and compHuman sample collection and analysis for mRNA quantification by the GTEx portal has previously been described . The meaM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 2.5 mM EDTA, 0.1% SDS, and 50 mM Tris-HCl, pH 8, with protease inhibitor cocktail . Homogenates were centrifuged at 15,000 rpm at 4°C for 20 min. The clear supernatant was stored at −70°C. Protein concentrations within each homogenate were determined with the Pierce Coomassie protein assay reagent . A 25 μg protein sample was run on 4–15% polyacrylamide gel  at 100 V for 1 h. Proteins and molecular weight markers  were transferred by electrophoresis onto an Immunobilon-P membrane  at 4°C and processed for immunodetection. Incubation of the membrane with blocking solution (3% non-fat milk in PBS for 1 h at RT) was followed with incubation in primary antibody  at 1 μg/mL dilution in blocking solution at 4°C overnight and washed three times. The membrane was next incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody  1:2,000 dilution for 2 h. A Lumigen ECL Ultra kit was used for detection. Bands were digitized on a Kodak ImageStation 440CF. The intensity of bands was quantitated with ImageJ software (NIH). After image capture, blots were stripped using Restore Western blot Stripping Buffer (Thermo Fisher Scientific) and reprobed with tubulin antibody (MilliporeSigma) as an internal control for sample loading.Proteins were isolated from mouse brain tissue with RIPA lysis buffer containing 150 m2 and 1 μL of varying half-log concentrations of inhibitor or a standard curve of 16β-hydroxytestosterone were added to designated wells and incubated at 37°C for 2 min. The substrate testosterone  and 10 μL of the NADPH generating system were added to all wells and incubated for 10 min at 37°C. The reaction was stopped by adding 100 μL of methanol containing 1 μM of the internal standards bucetin and diclofenac. The plates were sealed and placed in a −20°C freezer for 10 min, centrifuged at 5,700 rpm for 20 min at 4°C and the supernatants transferred for LC-MS/MS analysis.In 96-well plate format, 88.5 μL of human CYP46A1R bactosomes  in potassium phosphate buffer with MgClThe NADPH generating system was prepared by combining 1,500 μL of 200 mM glucose-6-phospate, 3,000 μL of 20 mM NADH, 60 μL of 1,000 U/mL glucose-6-phosphate dehydrogenase and 1,440 μL of 100 mM potassium phosphate at pH 7.4.Samples were injected onto a Waters Acquity UPLC with an Acquity UPLC HSS T3 1.8 μm, 2.1 × 50 mm column and triple quadrupole Xevo TQ MS detector using a gradient elution using mobile phase A of water with 0.1% formic acid and mobile phase B of acetonitrile with 0.1% formic acid. The gradient was maintained at 2% B for 0.05 min, then ramped linearly to 95% B in 0.95 min, then immediately returned linearly to 2% B in 0.50 min with a total runtime of 1.8 min. The reaction product 16β-hydroxytestosterone was monitored (parent m/z 305.1844 and daughter m/z 96.9690) and quantified. Dose response curves were generated by graphing 16β-hydroxytestosterone levels vs. inhibitor concentrations.2. 1 μL of varying half-log concentrations of inhibitor were added to designated wells and incubated for 5 min. Cholesterol (25 μM) was then added and the reaction was initiated using 1 mM NADPH. The reaction was conducted at 37°C for 30 min in a total incubation volume of 50 μL. After the incubation 30 μL of incubation mixture was added to a pre-made quench plate containing 30 μL 1 μM d7-24 hydroxy cholesterol in acetonitrile/methanol (50/50) to terminate the reaction. Subsequently, the plates were sealed and placed in a −20°C freezer for 10 min, centrifuged at 5,700 rpm for 20 min at 4°C and the supernatants transferred for LC-MS/MS analysis.In 384-well plate format, 0.5 μL of human CYP46A1 microsomes  were incubated for 5 min with 2-hydroxypropyl-β-cyclodextrin (10 mg/mL) and 100 mM potassium phosphate buffer (pH 7.4) containing 5 mM MgClSamples were injected onto a SCIEX 6500 QTRAP Mass Spectrometer with an Eclipse XDB-C18  column using a gradient elution using mobile phase A of water with 0.1% formic acid and mobile phase B of methanol:acetonitrile  at a flow rate of 600 μL/min at 50°C. The gradient was maintained at 80% B for 0.01 min, then ramped linearly to 100% B in 3 min, then returned linearly after 3.3 min to 80% B in 4.30 min with a total runtime of 11 min. The reaction product 24-hydroxycholesterol was monitored (parent m/z 385.0 and daughter m/z 367.0) and quantified in comparison to d7-24-hydroxycholesterol standard. Dose response curves were generated by graphing 24-hydroxycholesterol levels vs. inhibitor concentrations.ad libitum. The mice were acclimated for 5 days under these conditions before testing. The animals were randomly assigned to experimental groups; each mouse was used only once. At the time of the experiment mice were 5–6 weeks old. The experiment was approved by the Institutional Animal Care and Use Committee of Shanghai ChemPartner Co., Ltd.Male CD1 mice  were housed in plastic cages with metal covers  in groups of 5 under controlled conditions  with a standard rodent diet and water available Under the assay conditions, both 24S and 24R-hydroxycholesterol were detected in a single peak and quantified as total 24-hydroxycholesterol. Total 24-HC was measured using ester hydrolysis, liquid-liquid extraction, and measurement by LC-MS/MS. Brain or plasma samples were first diluted with PBS at a ratio of 3 mL of PBS to 1 g of tissue. Tissue homogenate was prepared using a bead mill type homogenizer (Spex-certiprep Genogrinder).The 50 μL sample with 5 μL of 24-hydroxycholesterol-d6 internal standard was added to 100 μL of 0.35 N KOH in methanol and heated to 80°C for 2 h. Hydrolyzed plasma or tissue homogenate (155 μL) was mixed with 200 μL of DI water and extracted with 1 mL of methyl-t-butyl ether. The organic layer was separated and taken to dryness under nitrogen at 50°C. The dry residue was reconstituted in 100 μL of 80% methanol in DI water. Reconstituted sample (5 μL) was injected on an ACE 3C18 0.5 mm × 1.0 mm HPLC column. Gradient elution was performed using an Eksigent LC200 HPLC system running a simple water:acetonitrile gradient from 70% acetonitrile to 95% acetonitrile over 4.75 min at a flow rate of 100 μL/min.Calibrators and assay quality controls were made by spiking 24S-hydroxycholesterol-d7 into control mouse plasma or brain homogenate and preparing them as samples. Detection of 24S-HC was accomplished using a Sciex Qtrap 5500 mass spectrometer running selected reaction monitoring of the analyte and internal standard. Ions were formed using a TurboV electrospray ion source operated in the positive ion mode. Sample concentrations were determined using the peak area ratio of analyte to internal standard and the least squares linear regression equation from the standard curve.Assay acceptance criteria for each LC-MS/MS run were ±20% accuracy compared to the nominal spiked concentration and ±20% CV. Endogenous quality controls made from control plasma or brain tissue were used to track longitudinal assay performance. Endogenous quality controls must reproduce within 3 SDs of the latest 20 measurements.ex vivo studies were approved by the Washington University Institutional Animal Care and Use Committee in accordance with international guidelines. Hippocampal slices were freshly prepared from the dorsal hippocampal region of postnatal day (P) 30–34 Harlan Sprague-Dawley male albino rats or C57Bl/6 mice  using glass electrodes filled with 2 M NaCl (5–10 MΩ resistance).At the time of study, slices were transferred individually to a submersion-recording chamber that was maintained at 30°C with continuous ACSF perfusion at 2 mL/min. Extracellular recordings were obtained from EPSPs were evoked using 0.1 ms constant current pulses through a bipolar stimulating electrode in the Schaffer collateral (SC) pathway. Responses were monitored throughout experiments using single stimuli every 60 s at half maximal intensity based on a control input-output (IO) analysis. After establishing a stable baseline for at least 10 min, long-term depression (LTD) was induced using 1 Hz low frequency stimulation (LFS) for 15 min (900 pulses), a stimulation protocol that reliably induces NMDAR-dependent LTD . IO curvp < 0.05 or better) between experimental variables. A two-way ANOVA with multiple comparison was used for assessment of compound 1 on 24S-HC at indicated timepoints .Electrophysiogical data were collected and analyzed using PClamp software . Data in the text are expressed as mean ± SEM. A two-tailed Student’s Human CYP46A1 expression, based on RNA-seq quantification, is preferentially expressed within the central nervous system, where transcripts are in excess of 100 per million while in most of the peripheral tissues they are less then 5 transcripts per million . The onlIn mouse, we used an antibody to quantify CYP46A1 protein expression. Similar to human, mouse CYP46A1 distribution displays brain region specific expression. CYP46A1 antibody was first confirmed to be immunoreactive against whole brain lysates from wildtype, but not CYP46A1 knockout mice, on western blot , reinforSince the presumed function of the CYP46A1 enzyme is to catalyze synthesis of 24S-HC  we also in vitro with IC50s of 26 and 37 nM, respectively  (p = 0.0027) in a 24S-HC sensitive manner (p = 0.006) . However, 24S-HC overcame the LTD deficit induced by compound 1 (p = 0.046) (p = 0.144)  , and we = 0.049) . Voricon= 0.049) , also de= 0.006) . The eme= 0.046)  but fail= 0.144) . Pharmac= 0.144) . Potentiin vitro CYP46A1 IC50 for at least 4 h  Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Shanghai ChemPartner Co., LTD., and the Washington University Institutional Animal Care and Use Committee.MP, AH, CZ, and SMe designed the study. YI, JD, and H-JS ran experiments. MP and SMe wrote the manuscript. AH, JD, SMi, and CZ edited the manuscript. MP and SMe analyzed the results. AH synthesized compounds 1 and 2. All authors contributed to the article and approved the submitted version.MP, AH, JD, and SMi were employees of Sage Therapeutics while this work was performed. Other authors were employed by Washington University in St. Louis School of Medicine. CZ was a member of the scientific advisory board for Sage Therapeutics and holds equity in Sage Therapeutics.


However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.The mitochondrial Ca Ca2+ is taken up by a channel known as mitochondrial Ca2+ uniporter (MCU), and once inside, it stimulates several dehydrogenases c) and optimally activated at high [Ca2+]c c c below the threshold [MICU1-deficient mitochondria of mammalian cells were found to be more sensitive to RuRed or Ru360 inhibition of Cachondria . This wa [Ca2+]c . This in [Ca2+]c ,27. Inte [Ca2+]c  as well  [Ca2+]c . Howevere tested . In thisof MICU2 . It is p [Ca2+]c , as it o [Ca2+]c , as the hreshold .T. cruzi plasma membrane is rich in ergosterol and GPI-anchored proteins  = dK × [(F − Fmin)/(Fmax − F)] was used to convert the raw fluorescence readings measured during mitochondrial Ca2+ transport assays into Ca2+ concentration levels, where dK is the dissociation constant, F is any given fluorescence value, Fmin is the lowest fluorescence reading after addition of 0.5 mM EGTA, and Fmax is the maximal fluorescence obtained after two sequential additions of 1 mM CaCl2. These additions were performed at the end of each trace. dK for Ca2+ indicator probes in our conditions was determined, according to Chweih et al. [Cells were collected by centrifugation at 1000× h et al. . Uptake T. cruzi epimastigotes (5 × 107 cells) were incubated at 28 °C in reaction buffer  containing 5 mM succinate, 0.2% BSA, 50 μM EGTA, and 5 μM safranine O, and the reaction was started with digitonin (50 μM). ADP (250 μM), carboxyatractyloside (1.5 μM), and FCCP (4 μM) were added to the medium at different time points. Fluorescence changes were monitored using the Hitachi F-7000 spectrofluorometer (excitation of 495 nm and emission of 586 nm).Estimation of mitochondrial membrane potential in situ was done spectrofluorometrically using the indicator dye safranine O, as described previously . Brieflyg for 7 min and washed twice with buffer A with glucose . Epimastigotes were resuspended to a final density of 1 × 109 cells/mL in BAG and kept on ice. Cells (1 × 108) were incubated at 28 °C in a 2 mL chamber containing buffer A with glucose (with or without 10 μM Ru265). Oligomycin (5 μg/mL) and FCCP (0.2 μM) were sequentially added. The oxygen consumption rates of epimastigotes were measured using a high-resolution respirometer . The equipment was calibrated as reported by its manufacturer. OCR was calculated as the negative time derivative of the oxygen concentration measured in the close respirometer chambers and expressed per milligram of protein.Cells were collected by centrifugation at 1000× g for 7 min and washed twice with buffer A with glucose . Epimastigotes were resuspended to a final density of 1 × 109 cells/mL in BAG and loaded with Rhod-2 AM . The cells were washed twice with buffer A with glucose and pretreated with or without 50 μM Ru265 for 30 min. Before each experiment, a 50 μL aliquot of epimastigotes (5 × 107 cells) was added to the BAG. Mitochondrial Ca2+ uptake occurred after the addition of 1 μM nigericin (Nig) and 1 μM ionomycin (Ion) to rapidly release Ca2+ from neutral and acidic compartments. The addition of 8 μM FCCP increased the release of mitochondrial Ca2+. Fluorescence changes were monitored in an F-7000 fluorescence spectrophotometer (Hitachi) with excitation at 552 nm and emission at 581 nm.Cells were collected by centrifugation at 1000× 5 cells/well in 7% CO2 in RPMI medium plus 10% fresh fetal bovine serum. After 24 h, the medium was removed, and the cells were washed twice with Hanks solution and prefixed for 5 min at 4 °C with 2% glutaraldehyde in phosphate-buffered saline (PBS). After fixation, the cells were immediately washed with PBS. Prior to incubation with the cells, the parasites were preincubated with 1 μM antimycin A or 10 μM Ru265. After 30 min of incubation at 37 °C, the parasites were seeded onto Vero cells (5 × 106 parasites/well). After 1 h of incubation at 37 °C, the coverslips were washed once with PBS and mounted onto glass slides in Fluoromount-G containing 15 μg/mL of 2-(4-aminophenyl)-1H-indole-6-carboxamidine (DAPI), which stains host and parasite DNA. Coverslips were viewed on an Olympus BX60 microscope to quantify the number of host cells that contained adherent parasites and the number of adherent parasites per cell in randomly selected fields. Three hundred host cells were counted per sample in four independent experiments.Gamma-irradiated (2000 rads) Vero cells were grown at 37 °C in 24-well plates on a 13 mm-diameter round glass coverslip at a density of 2 × 105 cells) were plated onto sterile coverslips in a 12-well plate and incubated overnight at 35 °C in 7% CO2 in RPMI medium plus 10% fresh fetal bovine serum. Tissue culture-derived trypomastigote collections were incubated at 4 °C overnight to allow amastigotes to settle from swimming trypomastigotes. Trypomastigotes from the supernatants of these collections were counted and used to infect the coverslips at a ratio of 50 parasites to 1 host cell. Prior to incubation with the cells, the parasites were preincubated with 1 μM antimycin A or 10 μM Ru265 for 30 min. At 4 h post-infection, coverslips were washed extensively with Hanks’ solution, followed by phosphate-buffered saline (PBS) at pH 7.4 to remove any extracellular parasites. Coverslips were fixed immediately in 4% paraformaldehyde in PBS (pH 7.4) at 4 °C for 30 min. Coverslips were washed once with PBS and mounted onto glass slides in Fluoromount-G containing 15 μg/mL of 2-(4-aminophenyl)-1H-indole-6-carboxamidine (DAPI), which stains host and parasite DNA. Coverslips were viewed on an Olympus BX60 microscope to quantify the number of host cells that contained intracellular parasites and the number of intracellular parasites per cell in randomly selected fields. Three hundred host cells were counted per sample in four independent experiments.Gamma-irradiated (2000 rads) Vero cells  for comparisons between more than two cell lines and two-way ANOVA with multiple-comparison tests for analyses of grouped data.Statistical analyses were performed with GraphPad Prism software version 8.4 . Reported values are means ± standard deviation (SD) from


Hes1 dimerization inhibitory activity of lindbladione (1) was discovered using our original fluorescent Hes1 dimer microplate assay. We also found that lindbladione (1) accelerates the differentiation of neural stem cells. We conducted the first total synthesis of lindbladione (1) via Heck reaction of 1-hexene-3-one 7 with iodinated naphthoquinone 12, which was provided by Friedel–Crafts acylation followed by Claisen condensation, in the presence of Pd (II) acetate. Careful deprotection of the benzyl groups of 13 successively provided lindbladione (1). Synthesized lindbladione (1) exhibited potent Hes1 dimer inhibition (IC50 of 2.7 μM) in our previously developed fluorescent Hes1 dimer microplate assay. Synthesized lindbladione (1) also accelerated the differentiation of C17.2 mouse neural stem cells into neurons dose dependently, increasing the number of neurons by 59% (2.5 μM) and 112% (10 μM) compared to the control. These activities are comparable to those of naturally occurring lindbladione (1) isolated from L. tublina.Lindbladione ( NSCs are self-renewing, multipotent cells that provide new neural cells such as neurons, astrocytes, and oligodendrocytes. Acceleration of NSC differentiation in the brain is an attractive approach to overcome neurodegenerative diseases. However, the development of regenerative medicine and small-molecule NSC activators has been slow due to the complex mechanism underlying the differentiation of NSCs into neural cells. In addition, internal NSCs are reportedly quiescent3.Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, affect many body activities, such as balance, movement, and talking. Millions of people worldwide are affected by these diseases, which severely compromise their quality of life. The discovery of neural stem cells (NSCs) in human adult brain4. Proneural bHLH factors, such as Ascl1 (formerly Mash1) and Neurog2, accelerate neurogenesis. Hairy and enhancer of split (hes) factors, such as Hes1, promote the self-renewal of NSCs and the generation of astrocytes, and they also suppress the expression of proneural genes. Recently, Kageyama et al. reported that oscillations in the concentration of bHLH factors directly control the fate of NSCs7. Hes1 and Ascl1 proteins oscillate with 2- to 3-h periodicity in active NSCs to activate cell proliferation. During cell fate selection, one bHLH factor is expressed in a sustained manner while expression of the others is repressed. In neurons, Ascl1 is expressed sustainably, whereas Hes1 expression is inhibited. However, in the adult brain, NSCs are in quiescent state, with high expression of Hes1. Inactivation of Hes1 and up-regulation of Ascl1 expression leads to an increase in neurogenesis7.The fate of NSCs, whether self-renewal or differentiation into neural cells, is controlled by basic helix-loop-helix (bHLH) transcriptional factorsAscl1 and other proneural genes to inhibit their expression  methods” using protein-immobilized beads16. Using Hes1 protein beads, agalloside, isomicromonolactam, inohanamine, and 4-O-ellagic acid were isolated as accelerators of the differentiation of NSCs into neurons15. Here, we report the first total synthesis of the Hes1 dimerization inhibitor lindbladione isolated from Lindbladia tubulina17. The attempted synthesis of lindbladione derivatives using a calculation approach is also reported.Because the Hes1 homodimer binds to the promoter region of 1) was considered to be obtained by the Heck reaction between 2-hydroxy-3-iodo naphthoquinone and α,β-unsaturated ketone, as in a previous report of naphthoquinone synthesis18. 2-Hydroxy-3-iodo naphthoquinone would be synthesized by Claisen condensation of the aromatic keto ester, followed by iodination. Friedel–Crafts acylation was selected to obtain the aromatic keto ester. Starting with trimethoxyphenylacetic acid 2, chlorination by thionyl chloride followed by addition of MeOH gave methyl ester 3  resulted in a decrease in yield (51%). Claisen condensation of keto ester 4 in the presence of sodium methoxide gave cyclized product 5 at moderate yield. 2-Hydroxy-3-iodo naphthoquinone 6 was obtained by iodination of 5 by the morpholine-I2 complex after 2 h19. The Heck reaction of 6 with 1-hexene-3-one 7 and 10 mol% Pd(OAc)2 in the presence of 5 eq. of K2CO3 gave the desired product 8 at 64% yield. Although deprotection of the methyl ether was examined under several conditions , all conditions failed to give lindbladione (1). Therefore, several protective groups were examined, including benzyl, acetyl, and methoxymethyl groups. However, Friedel–Crafts acylation did not proceed in all cases. Based on these results, the protective group was changed after the Friedel–Crafts acetylation.The results of retrosynthetic analyses are shown in Fig. 4 by 3 eq. of BBr3 gave a mixture of trihydroxy- and dihydroxyphenyl compounds at a 4:1 ratio , N-iodosuccinimide and CH2Cl2 reflux conditions gave 12 at 74% yield. The Heck reaction of 12 with 1-hexene-3-one 7 and 20 mol% Pd(OAc)2 in H2O-DMSO at 110 °C gave the benzyl-protected lindbladione 13.The deprotection of keto ester 2, BBr3, trifluoroacetic acid, AlCl3 and NaI, BF3 and Me2S, TiCl4) resulted in failure to obtain the deprotected compound. However, careful addition of BCl3  to 13 in 0.011 M CH2Cl2 at − 78 °C (low-concentration condition) and a short reaction time (10 min) gave the target product lindbladione (1) at quantitative yield. Extending the reaction to 20 min decreased the yield (58%) due to the instability of 1 under these conditions. Data for NMR spectra (1H and 13C) and high-resolution mass spectra were all identical with the values for naturally occurring 1.Next, we investigated deprotection of the benzyl group. Many conditions  interact with Lys74 and Lys77. Two phenolic hydroxy groups of 1 were found close to Lys58. Interestingly, CH/π interaction between 1 and Lys77 was also predicted. Next, we performed in silico screening of derivatives of 1. R substituents of 1 were screened from a linker database containing 3408 units and 26 commercially available compounds  inhibited Hes1 dimer formation dose dependently, with an IC50 value of 2.7 μM, comparable to that of naturally occurring 1 (3.7 μM). The protected compounds 5, 6, and 8 did not exhibit inhibitory activity, which suggested that the phenolic hydroxy groups are important for this activity . Indeed, there was lower energy conformation of 14 with Hes1 . However, the difference of molecular weight might result in lower energy. And also, the calculation results showed compound 14 binds to Hes1 with several forms (five main forms). The highest (unstable) energy was − 4.6542 kcal/mol, this is almost same as that of lindbladione (1) . This might be one of the reasons why Hes1 dimer inhibition of 14 was not as potent as 1. In addition, the hydrophobic interaction of methyl on the side chain of 1 seems to be important. However, methyl ketone in 14 might be not efficient for suitable hydrophobic interaction.The inhibition of Hes1 dimer formation was investigated using our original Hes1 dimer plate assay Fig. 8. We presed Fig. . Synthet1 on NSC differentiation was examined , retinoic acid21 , or synthetic 1. After incubation for 4 days, the cells were immunostained , and the number of neurons was determined. Synthetic 1 accelerated the differentiation of NSCs into neurons by 59% at 2.5 μM and 112% at 10 μM. As naturally occurring 1 induced a 25% increase in the number of neurons at 2.5 μM14, the results for synthetic 1 were comparable to those of naturally occurring 1.Next, the effect of synthetic ned Fig. . Mouse N1), was accomplished. Synthetic 1 exhibited comparable Hes1 dimerization inhibition in our original Hes1 dimerization plate assay and accelerated NSC differentiation comparable to isolated 1. As lindbladione (1) exhibited good selectivity against other basic helix-loop-helix transcriptional factors, such as the interaction between TCF and β-catenin  spectra. JASCO FT/IR-4700 spectrometer was used for recording IR spectra. JEOL AccuTOF LC-plus JMS-T100LP mass spectrometer was used for obtaining HRMS spectra. JASCO an LC-2000 Plus series were used for high-performance liquid chromatography (HPLC).8 , and wells were incubated with 50 μL of Cy3-labeled-Hes1 in NET-N buffer  containing 0.05% Nonidet P-40, ca. 7 mg/L) for 21 h at 4 °C. To remove extra the Cy3-proteins, the wells were washed twice with 200 μL of PBST. To the wells was added each compound solution  and the plates were incubated for 1 h at room temperature (r.t.) in the dark. The wells were washed twice with 200 μL of PBST, then dried under reduced pressure for 1 h in the dark. The Cy3 dye was excited at 544 nm and emission was monitored at 590 nm using a microplate reader . The assays were carried out in three individual wells, and the mean value and SD were calculated.Hes1-bound microplate wells were constructed as reported before5 cells/mL) in proliferation medium were seeded on cover glass (Matsunami Glass) coated with fibronectin/laminin and poly-L-lysine. Cells were washed with D-MEM after incubation for 12 h, then medium was changed to differentiation medium to initiate differentiation of C17.2 cells with individual compounds for 4 days. Immunofluorescence staining method was reported before11. Proliferation medium: DMEM  supplemented with 10% fetal bovine serum (BioWest) and 5% horse serum (Gibco), differentiation medium: Neurobasal Medium (Gibco) with 2% B-27 without vitamin A (Gibco) and 1% antibiotic–antimycotic (Gibco).C17.2 cell line was purchased from European collection of cell cultures. C17.2 cells (2 × 101) were constructed using Build in MOE with standard geometric parameters. Then, fragment-based drug design was performed with lindbladione (1) as the parent nucleus to generate 3434 analogs using Fragment-based drug design in MOE. Lindbladione (1) and 3434 analogs were minimized using Energy Minimize in MOE with the Amber force field until the root-mean-square (rms) energy gradient was less than 0.001 kcal mol−1 Å−1. The molecular docking simulations, lindbladione (1) and 3434 analogs for Hes1 homodimer domain, were performed using the rigid methods in MOE-Dock.Hes1 homodimer domain was constructed based on the structure of HLH domain taken from the Protein Data Bank (PDB) (PDB code: 2MH3) by using MOE . Water molecules in the crystal structure were removed. All hydrogen atoms were added and Amber all-atom charges were assigned for the whole protein. The initial 3D structures of lindbladione . A Supplementary Information


Shigella IpaH9.8 and the LRR of IpaH9.8 in complex with the substrate of human guanylate-binding protein 1 (hGBP1). A hydrophobic cluster in the C-terminus of IpaH9.8LRR forms a hydrophobic pocket involved in binding the NEL domain, and the binding is important for IpaH9.8 autoinhibition. Substrate-binding destabilizes the hydrophobic cluster by inducing conformational changes of IpaH9.8LRR. Arg166 and Phe187 in IpaH9.8LRR function as sensors for substrate-binding. Collectively, our findings provide insights into the molecular mechanisms for the actication of IpaH9.8 in autoinhibition mode 2.IpaH enzymes are bacterial E3 ligases targeting host proteins for ubiquitylation. Two autoinhibition modes of IpaH enzymes have been proposed based on the relative positioning of the Leucine-rich repeat domain (LRR) with respect to the NEL domain. In mode 1, substrate-binding competitively displaces the interactions between theLRR and NEL to relieve autoinhibition. However, the molecular basis for mode 2 is unclear. Here, we present the crystal structures of  LRR–hGBP1. They find that substrate-binding destabilizes the hydrophobic cluster to relieve the autoinhibition of IpaH9.8. This study provides insights into the mechanisms underlying substrate-induced activation of IpaH9.8.Ye, Xiong et al. present crystal structures of bacterial E3 ubiquitin ligase IpaH9.8 and IpaH9.8 In the ubiquitin-proteasome system (UPS), the 76-amino acid ubiquitin (Ub) is conjugated to lysine residues in target proteins. Ubiquitination is mediated by a three-enzyme cascade consisting of the Ub-activating enzyme (E1), the Ub-conjugating enzyme (E2), and the Ub-protein ligase (E3)6. E3s play a crucial role in this cascade for their precise control of both the efficiency and substrate specificity in ubiquitination7. E3s can be grouped into three classes: the RING  E3s, the HECT (Homologous to E6AP C-terminus) E3s, and the RBR (Ring-Between-Ring) E3s8. These three classes of E3s use two distinct strategies to transfer Ub. The RING E3s, which are characterized by their RING or U-box catalytic domain, directly transfer Ub from an E2 to the target substrate11. In contrast, the HECT and RBR E3s, both of which utilize active site cysteines, catalyze ubiquitination in a two-step strategy: (1) transfer Ub from the E2–Ub to the E3 catalytic cysteine residue in a transthiolation reaction and (2) then transfer Ub from the E3–Ub to the substrate lysine (or the N terminus) in a subsequent aminolysis reaction14.Ubiquitination, as one of the most important protein post-translational modifications, regulates a multitude of biological processes in eukaryotic cells15. Host cells can utilize the UPS to activate innate immune responses against bacterial invasion, but invasive pathogens can also use the UPS against the host in order to facilitate infection19. The IpaH family is a group of conserved effectors that are secreted by Shigella, Salmonella, and other Gram-negative bacteria through type III secretion systems21. IpaH proteins function as E3 ligases and subvert the host UPS pathway to suppress the host inflammasome and innate immune responses22. These enzymes contain an N-terminal type III secretion signal sequence, a leucine-rich repeats (LRR) domain involved in substrate recognition, and a C-terminal conserved novel E3 ligase (NEL) domain23. Although the NEL domain has little sequence or structural homology to known HECT E3s, IpaH enzymes possess E3 ligase activity that is dependent on a catalytic cysteine in the NEL domain, which facilitates Ub transfer from E2 to the substrate in a manner similar to HECT E3s25.Ubiquitination plays important roles in pathogenic infection27. Two distinct autoinhibition modes have been proposed based on the crystal structures of SspH2 (mode 1) and IpaH3 (mode 2)29. In SspH2 structure (mode 1), the concave surface of the LRR domain orients toward the NEL domain, whereas in IpaH3 structure (mode 2), the concave surface is oriented in an opposite direction relative to the NEL domain27. Binding of the substrate to the LRR domain could activate the IpaH in both autoinhibition modes30. Previously, the crystal structure of SspH1LRR-PKN1HR1b revealed that substrate-binding competitively displaces interactions between the LRR and NEL domains, thereby relieving autoinhibition in mode 1 (ref. 31). However, the mechanistic details that explain how substrate binding triggers IpaH activation in mode 2 are missing.Autoinhibition is an important regulatory mechanism by which IpaH enzymes prevent self-degradation from the host UPS or activation of the host innate immune response via formation of free polyUb chainsShigella flexneri during infection23. IpaH9.8 targets Ste7 for degradation in order to hijack the host UPS and subsequently inhibit mitogen-activated protein kinase (MAPK)-dependent signaling pathways in yeast23. Additional studies have shown that IpaH9.8 plays an important role in modulating host inflammatory responses by ubiquitinating NF-κB essential modulator (NEMO) and then promoting NEMO degradation32. Recently, it has been reported that IpaH9.8 targets human guanylate-binding protein 1 (hGBP1) for ubiquitination and degradation in order to suppress host defense in cells35. As the best-characterized member of the GBP family, hGBP1 is a GTPase with both antiviral and antibacterial activities that is encoded by an interferon-activated human gene36. hGBP1 consists of a large GTPase domain (LG) at the N-terminus, an α-helical middle domain , and an α-helical GTPase effector domain  at the C-terminus39. As one member of the dynamin superfamily, hGBP1 greatly increases GTP hydrolysis activity upon oligomerization41. When hGBP1 binds to different guanine nucleotides or transition state analogs, it adopts different oligomer/monomer states45. Previous biochemical work has revealed that the recognition of hGBP1 by IpaH9.8 is independent of the nucleotide-bound state of hGBP1 (ref. 34). However, there is a dearth of direct structural evidence supporting this concept.IpaH9.8 is one of the most well-studied members of the IpaH family and is secreted by 46). In this study, we solved the crystal structure of the near full length IpaH9.8, and observed that the architecture of IpaH9.8 is similar to IpaH3 in autoinhibition mode 2. Based on the structure of IpaH9.8, we investigated the importance of a hydrophobic cluster in the C-terminal region of IpaH9.8LRR for IpaH9.8 autoinhibition. Then, to study the activation mechanism of IpaH9.8 in mode 2, we solved the crystal structure of the LRR domain of IpaH9.8 (IpaH9.8LRR) in complex with full-length hGBP1. By comparing the structures of IpaH9.8LRR in apo and substrate-bound forms, we illuminated the mechanisms by which substrate activates IpaH9.8 via induction of conformational changes in IpaH9.8LRR. Further structural analysis revealed that two residues located in the concave surface of IpaH9.8LRR function as sensors for substrate binding. Finally, we described the molecular basis for Ub transfer from NEL to substrate after activation. Collectively, this study reveals a substrate-induced activation mechanism of IpaH9.8 in mode 2.IpaH9.8 is 82% identical to IpaH3, suggesting that the IpaH9.8 might autoinhibit its enzymatic activity similarly to IpaH3 in mode 2  was crystallized in non-reducing conditions and reported to be an inactive, likely an artificial structure47. In the IpaH9.8NEL-iso structure, the active cysteine from two different asymmetric units formed a disulfide bond and caused α8 and α9 to extend into a long straight helix  showed a “helix-loop-helix” fold, with the loop region between α8 and α9 being flexible and not observable in electron density map  was determined by crystallization and X-ray diffraction at a 2.75 Å resolution Fig. . The cryces Fig. , right. ces Fig. , left30.lix Fig. . Howeverlix Fig. , suggestmap Fig. . Compareain Fig. . Moreoveain Fig.  is consiNEL-FL is similar to IpaH3 in mode 2  at 3.6 Å was reported49. This allowed for a more systematic structural analysis of the IpaH9.8LRR–substrate complex under different conditions.To obtain the complex structure of IpaH9.8–substrate, we initially attempted to crystalize the full-length (or near full-length) IpaH9.8 in complex with the full-length hGBP1 but did not succeed. Crystals were obtained for the complex of the IpaH9.8 LRR domain and the full-length hGBP1, and the structure of the IpaH9.82 Å Fig. . The comLRR–hGBP1 and the IpaH9.8LRR–hGBP1LG-MD complex structures is within the MD domain of hGBP1 . In the IpaH9.8LRR–hGBP1 complex, the guanine-cap was visible and revealed a similar orientation to other nucleotide-bound structures of hGBP1 . These observations provide direct structural evidence that the recognition of hGBP1 by IpaH9.8 is independent of its nucleotide-bound state34.The structural comparison reveals that the two complex structures correspond to one another in how IpaH9.8lex Fig. . FurtherBP1 Fig. , and theBP1 Fig. . This diure Fig. . In addiLRR–hGBP1, named IpaH9.8LRR-Sub; in IpaH9.8LRR–hGBP1LG-MD, named IpaH9.8LRR-Sub’; in IpaH9.8FL, named IpaH9.8LRR-FL; and in isolated IpaH9.8LRR (ref. 50), named IpaH9.8LRR-iso. Comparison of these LRR domains unmasked great conformational differences in the LRR-CT during substrate engagement  rotated ~10 Å and ~20 Å and translates ~5 Å and ~10 Å toward the convex side with respect to IpaH9.8LRR-FL and IpaH9.8LRR-iso, respectively  were with different angles between IpaH9.8LRR-FL and IpaH9.8LRR-Sub  caused a considerable reduction in hGBP1 ubiquitination  as a wild-type enzyme  completely abolished ubiquitination activity in Ipah9.8 for hGBP1, but did not affect the polymerization of Ub chain  and the linker of SspH1  recovered the activity of IpaH9.8 for hGBP1 ubiquitination  In apo form, the hydrophobic cluster in the LRR-CT of IpaH9.8 forms a hydrophobic pocket to bind NEL and inhibits its catalytic activity  were synthesized by GENEWIZ, China. The cDNA of human UbcH5b and mouse UBA1 were provided by Prof. Feng Rao  and Prof. Naixia Zhang , respectively. IpaH9.8, IpaH9.8∆N21(21–545), IpaH9.8LRR (21–244), IpaH9.8NEL (254–545), hGBP1, Ub, and UbcH5b were cloned into pET28M, an Escherichia coli expression vector with the original N-terminal thrombin protease site replaced by a Tobacco Etch Virus protease site. Mouse UBA1 was cloned into pET28a. All mutants were introduced using PCR-based mutagenesis and confirmed by sequencing.The wild-type (WT) gene of E. coli strain BL21 (DE3) Codon Plus (Stratagene). Large-scale cultures were grown in LB medium at 37 °C until the optical density at 600 nm reached ~0.6. Overexpression of proteins was then induced by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.25 mM. After growing at 18 °C for 16 h, the cells were harvested by centrifugation at 4500×g for 30 min at 4 °C. The cell pellets were washed twice with lysis buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 0.5 mM dithiothreitol, and then stored at −80 °C until use.Protein constructs for crystallization and in vitro ubiquitination activity assays were expressed in g for 30 min at 4 °C. The filtered supernatant was then loaded onto a Ni-affinity column  pre-equilibrated with lysis buffer using an AKTA pure system . The sample was eluted with a gradient of 0–500 mM imidazole in lysis buffer. Proteins for crystallization were further purified by anion exchange using a 5-mL HistrapTM Q column , and subsequently loaded onto a HiLoad 16/60 Superdex 200  pre-equilibrated with buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM dithiothreitol. Proteins used for in vitro ubiquitination activity assays were directly subjected to gel filtration by Superdex 200 or Superdex 75 columns. The peak fractions were concentrated using an Amicon Ultra-15 column, snap frozen in liquid nitrogen, and stored at −80 °C. All purification steps were performed at 4 °C and the eluted proteins were examined using SDS-PAGE.For the purification of the target proteins, the harvested cell pellets were resuspended in lysis buffer that was supplemented with 0.1 mg/mL DNase (Sigma), 0.5 mg/mL lysozyme (Sigma), and a protease inhibitor cocktail , and disrupted by sonication. The cell lysate was centrifuged at 40,000×IpaH9.8 (22-537) was concentrated to 25 mg/mL and the initial crystallization screening was performed using a sitting-drop vapor-diffusion method with a series of crystallization kits from Molecular Dimensions  in 96-well plates. Drops were prepared by mixing 0.75 µL of both protein solution and reservoir solution together, followed by equilibration with 75 µL of reservoir solution at 20 °C. Appropriate crystals were obtained within 1 week at 20 °C using a reservoir solution containing 2.5 M sodium chloride and 0.1 M potassium/sodium phosphate buffer (pH 6.0).LRR (22–244) and hGBP1 (full-length) complex, 0.3 mM IpaH9.8LRR was mixed with 0.3 mM hGBP1 and 5 mM GDP. Sitting drops screens were set as above. The first crystals could be observed after 3 days. After optimizing the conditions, appropriate crystals were obtained within 1 week at 20 °C using a reservoir solution containing 3.5 M sodium chloride and 0.1 M sodium citrate buffer (pH 6.5).Prior to applying the crystallization screens for the IpaH9.8∆21 and the IpaH9.8LRR and hGBP1 complex were collected using the beamline BL17U at SSRF  at 0.97 Å wavelength and 100 K temperature. All data sets were indexed, integrated, and scaled using the XDS program suite62. All data collection statistics are summarized in Table All crystals were soaked in a cryoprotectant solution containing 20% (v/v) ethylene glycol and then snap frozen in liquid nitrogen prior to shipment. Diffraction data from the crystals of IpaH9.8Phaser in the Phenix program package63. The structure of IpaH9.8LRR (PDB: 5B0T) was used as the search model. Partial density was visible for IpaH9.8NEL. Sculptor in the Phenix program suite was used to create a model of IpaH9.8NEL using IpaH3NEL (PDB: 3CVR) as a base structure, which was placed into the available density. Several rounds of refinement were performed using the phenix.refine program in the Phenix program suite, alternating with manual fitting and rebuilding based on 2Fo − Fc and Fo − Fc electron density using COOT64.The structure of IpaH9.8 was determined using the molecular replacement method with the program LRR and hGBP1 complex was determined using the structure of IpaH9.8LRR (PDB: 5B0T) and hGBP1 (PDB: 1DG3) in a molecular replacement method63. Subsequently, the GDP molecular structure was constructed using the 2Fo − Fc and Fo − Fc electron densities. The model was refined via iterative rounds of refinement and rebuilding using Phenix, Refmac5, and COOT65.The structure of the IpaH9.866 are shown in Table Pymol or Chimera68. The electron density maps were constructed in Pymol from 2Fo − Fc maps contoured at the sigma level as indicated.Two crystal structures determined in this study display Ramachandran statistics with 91.59% and 91.46% of residues in the most favored regions and 8.19% and 8.15% of residues in the allowed regions of the Ramachandran diagram, respectively. The final refinement statistics and geometry of both crystal structures defined by MolProbity2, 5 mM ATP, and 2 µM hGBP1. Reactions were initiated by the addition of ATP. Sampled time points were taken by adding 20 µL of the reaction mixture to 20 µL of reducing SDS-PAGE load dye, followed by boiling prior to electrophoresis. SDS-polyacrylamide gels were loaded with 20 µL of sample, followed by electrophoresis, Coomassie staining, and imaging on a Bio-Rad Gel Doc (Bio-Rad). For immunoblot analysis, SDS-polyacrylamide gels were loaded with 5 µL of sample, followed by electrophoresis, transfer to a PVDF membrane, and incubated with specific primary antibodies as indicated. Ubiquitination assays for the polymerization of free Ub chains were performed as above but without hGBP1 and detected by Coomassie staining. Finally, the signals were visualized by the chemiluminescence system (Perkin Elmer). These experiments were conducted three times. The uncropped and unprocessed western blotting images were show in Supplementary Fig. Ubiquitination assays for substrate were performed in PBS buffer (pH 7.4) at 25 °C using 0.25 µM E1 (mouse UBA1), 2 µM E2 (UbcH5b), 0.5 µM E3, 50 µM Ub, 5 mM MgClB factor of Cα atoms were calculated by Eq. . Purified IpaH9.8-WT and IpaH9.8–R166A were titrated into fluorescently labeled hGBP1 and incubated for 30 min at room temperature before MST scans were performed using a Monolith NT. Automated device (NanoTemper Technologies). Binding curves were fit using NT Analysis Software. The results revealed that IpaH9.8 mutant R166A displayed similar curves to the wild-type IpaH9.8  were dialyzed in 10 mM sodium phosphate buffer at pH 7.5 and at 4 °C overnight before measurement. The CD spectra were obtained from a wavelength region of 190–260 nm, taking the average of four scans. The molar ellipticity per residue was calculated by (h) = h / (10n × c × l). Here, h is the CD signal in mdeg, n the number of residues, c the concentration in mol/L, and l the length of the cuvette path (cm). The sample concentrations were measured using a Quant-iTTM Protein Kit (Invitrogen) three times, and the average values were applied to calculate the molar ellipticity per residue. The result showed that all IpaH9.8 mutants display the same curves with the wild-type IpaH9.8  under a NEach experiment for ubiquitination assay was repeated three times, and sample sizes and numbers are indicated in detail in the Methods section.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


Shewanella vesiculosa HM13, a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel, is able to produce a large amount of EMVs. S. vesiculosa HM13 has been found to include a cargo protein, P49, in the EMVs, but the entire mechanism in which P49 is preferentially included in the vesicles has still not been completely deciphered. Given these premises, and since the structural study of the components of the EMVs is crucial for deciphering the P49 transport mechanism, in this study the complete characterization of the lipooligosaccharide (LOS) isolated from the cells and from the EMVs of S. vesiculosa HM13 grown at 18 °C is reported. Both lipid A and core oligosaccharide have been characterized by chemical and spectroscopic methods.Bacterial extracellular membrane vesicles (EMVs) are membrane-bound particles released during cell growth by a variety of microorganisms, among which are cold-adapted bacteria.  Shewanellalivingstonensis NF22T, a psychrotolerant Antarctic bacterium 3[C12:0(3-OH)][C13:0]2 .Both glycoforms were hexa-acylated, with a difference of 80 Da between the two clusters, each corresponding to a phosphate group. Variations of fatty acid length are responsible for the 14 Da mass differences within each cluster. In particular, to the ion at 4OH was performed. This procedure obtained a lipid A devoid of acyl and acyloxacyl esters, leaving intact acyl and acyloxacyl amides. The negative ions MALDI-TOF spectra of the samples (m/z = 1119.65 was attributed to a tri-acylated bis-phosphorylated lipid A species with two amide-linked 13:0(3-OH) residues, one of which is substituted at O-3 by a secondary 13:0 acyl chain . The differences of 14 Da less can be attributed to a C12:0 as secondary fatty acid as well as to a C12:0(3-OH) instead of a C13:0(3-OH) as primary fatty acid residues. Similarly, the additional 14 Da can be due to a combination of two C13:0(3-OH) residues and one C14:0, as well as a C14:0(3-OH) instead of a C13:0(3-OH) as a primary fatty acid.In addition, the mass spectra of the entire LOSs revealed that phosphoethanolamine substituted the lipid A in both structures . To dete samples b indicatShewanella pacifica [Shewanella species has never been described.Differences in the amount of the double phosphorylated species in comparison with the monophosphorylated ones in both lipid A spectra can be due to a partial hydrolysis of the phosphate group during the acetic acid treatment. Indeed, there are no differences in the relative amount of the intact lipid A species . Taking pacifica , we concO-acylated by mild hydrazinolysis and then de-N-acylated by strong alkaline hydrolysis [1H, 1H DQF-COSY, 1H, 1H TOCSY, 1H, 1H ROESY, 1H, 13C DEPT-HSQC, 1H, 13C HMBC and 1H, 31P HMBC). The 1H NMR spectra of the LOS carbohydrate backbone from the EMVs displayed the same eight anomeric signals already found for the oligosaccharide from cells grown at 4 °C . The culture was centrifuged at 6800× g for 10–20 min at 4 °C to pellet the cells, which were used for LOS analysis after washing with Dulbecco’s phosphate buffered saline. The culture supernatant was centrifuged again at 13,000× g for 15 min at 4 °C. The supernatant obtained was filtrated with 0.45-µm membrane filter. The filtrate was concentrated with MinimateTM Tangential Flow Filtration system equipped with a 500K Minimate capsule with Omega membrane  at 4 °C. The concentrate was ultracentrifuged at 100,000× g for 2 h at 4 °C to collect EMVs for LOS analysis.A rifampin-resistant mutant of osa HM13  was growDried cells (3.8 g) and EMVs (202 mg) were extracted using the PCP method . The LOSPolyacrylamide gel electrophoresis (PAGE) was performed using the system of Laemmli  with sod3OH  at 80 °C for 16 h. The mixture was extracted three times with hexane. The hexane phases containing the fatty acids methyl esters were directly analyzed by GC-MS. The methanol layers, containing the methyl glycosides were dried and then acetylated with acetic anhydride in pyridine at 100 °C for 30 min.The monosaccharides were analyzed as acetylated methyl glycosides by GC-MS . BrieflyAll the samples were analyzed on an Agilent Technologies gas chromatograph 6850A equipped with a mass selective detector 5973N and a Zebron ZB-5 capillary column . Acetylated methyl glycosides were analyzed using the following temperature program: 140 °C for 3 min, then 140→240 °C at 3 °C/min. The temperature program for methyl esters of fatty acids was the following: 140 °C for 3 min, then 140→280 °C at 10 °C/min, and finally 280 °C for 20 min.O-acylated LOS. The pellet was recovered after centrifugation at 4 °C and 7000 rpm for 30 min, washed three times with acetone, dissolved in water, and lyophilized (23 mg) [O-deacylated LOS (23 mg) was submitted to a reaction with 4 M KOH (1.0 mL) for 16 h at 120 °C. The reaction mixture was neutralized with 2 M HCl (until pH 6) and extracted with CHCl3 three times. The aqueous phase was desalted on a Sephadex G-10 column . The eluted fraction was then lyophilized (6.9 mg).The LOS sample from the EMVs (32.9 mg) was dried over phosphorus anhydride in a vacuum chamber and then treated with hydrazine (1.75 mL) at 37 °C for 2 h. Cold acetone was added to precipitate the de- (23 mg) . The O-d3CN in water (25 mg mL−1) was used as the matrix. The samples were desalted on a Dowex 50WX8 (H+ form) and dissolved in 2-propanol/water with a 1:1 ratio. The spectra were calibrated and processed under computer control by using Data Explorer software v.0.2.0 . Mass accuracy was found to be better than 170 ppm.MALDI-TOF mass spectra were acquired on an ABSCIEX TOF/TOF™5800  mass spectrometer equipped with an Nd:YLF laser with a λ of 345 nm, a < 500-ps pulse length, and a repetition rate of up to 1000 Hz. Approximately 2000 laser shots were accumulated for each spectrum. The calibration of the mass spectra was obtained with a hyaluronan oligosaccharides mixture. A solution of 2,5-dihydroxybenzoic acid (DHB) in 20% CH1H and 13C NMR spectra were recorded at 298 K with a Bruker Avance 600 MHz equipped with a cryoprobe. The chemical shifts were measured in D2O using sodium 3-trimethylsilyl--propanoate  as an internal standard for the protons. 1,4-dioxane (δC 67.40 ppm) was used for the carbon atoms. All 2D homo- and hetero-nuclear experiments  were performed as already reported [1H,31P HMBC experiment was recorded on a Bruker DRX-400 spectrometer as already reported [1D and 2D reported . The 1H,reported .S. vesiculosa HM13 were fully characterized. The characterization revealed the same structure for the cells and EMVs. Finally, no differences were found with respect to the LOS between the cells grown at 4 °C and 18 °C. Cold-adapted bacteria have been examined for the EMVs’ production and their LPSs’ content [Porphyromonas gingivalis the lipid A is less acylated in EMVs compared with cells [Pseudomonas aeruginosa, where two different LPSs have been identified, one of them is enriched in the EMVs relative to the cells [Bacteroides fragilis, the comparison of cellular lipid A and EMVs revealed that they were identical [In this study, the LOSs isolated from both the cells and EMVs of  content . Howeverth cells . In Pseuhe cells . Finallydentical .S. vesiculosa HM13 is capable of embedding a large quantity of P49 cargo protein into its EMVs, although it was also found in its cellular fraction [S. vesiculosa HM13 that have defects in biosynthesis of LOS are under investigation to understand if LOS-P49 interaction is necessary for the loading of P49 into the EMVs.fraction . To go dfraction . Mutants


Ganoderic acid A (GAA) is usually used to prevent cancers or other diseases, which make it likely to be used with other drugs metabolized by cytochromes P450. This study investigates the effect of GAA on eight major cytochrome P450 isoforms in human liver microsomes.in vitro using human liver microsomes (HLMs) with specific substrates for the CYPs, and the enzyme kinetic parameters were calculated. The effects of GAA (100 μM) on eight human liver CYP isoforms  were investigated 50IC values of 15.05, 21.83, and 28.35 μM, respectively. Additionally, GAA was not only a non-competitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2D6 and 2E1, with Ki values of 7.16, 10.07, and 13.45 μM. Meanwhile, the inhibition of CYP3A4 was time-dependent, with the I/KinactK value of 7.91/0.048 μM/min. The results showed that GAA inhibited the activity of CYP3A4, 2D6, and 2E1, but did not affect other isoforms. The inhibition of CYP3A4, 2D6, and 2E1 was concentration-dependent with in vitro study indicated that GAA has the potential to result in drug-drug interactions with other drugs metabolized by CYP3A4, 2D6, and 2E1. Further clinical studies are needed for the identification of this interaction. The  Cytochrome P450 enzymes (CYPs) are one of the most important phase-I enzymes that participate in the metabolism of most clinical drugs in liver and intestine. The status of CYPs might influence the pharmacokinetics of co-administrated drugs and result in adverse effects, which would contribute to costly and late failures of drug development  Karst (Ganodermataceae), also called Ling Zhi in China, has been widely used in traditional Chinese medicine for more than 2000 years to promote health and longevity, as it has been reported to have anticancer and many other beneficial properties , testosterone (CYP3A4), coumarin (CYP2A6), chlorzoxazone (CYP2E1), dextromethorphan (CYP2D6), diclofenac (CYP2C9), S-mephenytoin (CYP2C19) and paclitaxel (CYP2C8), we clarified the effect of GAA on the activity of CYPs, and the inhibition model was determined by the enzyme kinetic studies.In addition to the medicinal application of d-Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, corticosterone (≥98%), NADP+, phenacetin (≥98%), acetaminophen (≥98%), 4-hydroxymephenytoin (≥98%), 7-hydroxycoumarin (≥98%), 4′-hydroxydiclofenac (≥98%), sulfaphenazole (≥98%), quinidine (≥98%), tranylcypromine (≥98%), chlorzoxazone (≥98%), 6-hydroxychlorzoxazone (≥98%), paclitaxel (≥98%), 6β-hydroxytestosterone (≥98%), clomethiazole (≥98%), and furafylline (≥98%) were obtained from Sigma Chemical Co. Montelukast (≥98%) was obtained from Beijing Aleznova Pharmaceutical . Coumarin (≥98%), diclofenac (≥98%), dextromethorphan (≥98%), and ketoconazole (≥98%) were purchased from ICN Biomedicals. Pooled HLMs were purchased from BD Biosciences Discovery Labware. All other reagents and solvents were of analytical reagent grade.Ganoderic acid A (≥98%) and testosterone (≥98%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products . The chemical structure of GAA is shown in O-deethylation for CYP1A2, testosterone 6β-hydroxylation for CYP3A4, coumarin 7-hydroxylation for CYP2A6, chlorzoxazone 6-hydroxylation for CYP2E1, dextromethorphan O-demethylation for CYP2D6, diclofenac 4′-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19, and paclitaxel 6α-hydroxylation for CYP2C8. All incubations were performed in triplicate, and the mean values were utilized. The typical incubation systems contained 100 mM potassium phosphate buffer (pH 7.4), NADPH generating system , the appropriate concentration of HLMs, a corresponding probe substrate and hispidulin (or positive inhibitor for different probe reactions) in a final volume of 200 μL.As shown in The concentration of GAA was 100 μM, and the positive inhibitor concentrations were as follows: 10 μM furafylline for CYP1A2, 1 μM ketoconazole for CYP3A4, 10 μM tranylcypromine for CYP2A6, 50 μM clomethiazole for CYP2E1, 10 μM quinidine for CYP2D6, 10 μM sulfaphenazole for CYP2C9, 50 μM tranylcypromine for CYP2C19, 5 μM montelukast for CYP2C8. Probe substrates, positive inhibitors  and GAA were dissolved in methanol, with a final concentration of 1% (v/v), and 1% neat methanol was added to the incubations without inhibitor. The final microsomal protein concentration and incubation times for the different probe reactions are shown in 50IC). iK values were obtained by incubating various concentrations of different probe substrates  in the presence of 0–50 μM GAA.GAA (100 μM) was used to initially screen for its direct inhibitory effects towards different human CYP isoforms. For the CYP isoforms whose activities were strongly inhibited, secondary studies were performed to obtain the half inhibition concentration  was pre-incubated with HLMs (1 mg/mL) in the presence of an NADPH-generating system for 30 min at 37 °C. After incubation, an aliquot (20 μL) was transferred to another incubation tube  containing an NADPH-generating system and probe substrates whose final concentrations were approximate to KI and inactK values for the inactivation of CYP3A4, the incubations were conducted using higher probe substrate concentrations  and various concentrations of GAA (0–50 μM) after different preincubation times (0–30 min), with a two-step incubation scheme, as described above.To determine the maxV and mK. Inhibition data from the experiments that were conducted using multiple compound concentrations were represented by Dixon plots, and inhibition constant (iK) values were calculated using non-linear regression according to the following equation:iK is the inhibition constant, S is the concentration of the substrate, and mK is the substrate concentration at half the maximum velocity (maxV) of the reaction. The mechanism of the inhibition was assessed using the Lineweaver-Burk plots and the enzyme inhibition models. All data are represented as Mean ± SD. The data comparison was performed using Student’s t-test and performed using IBM SPSS statistics 20 (SPSS Inc.).The enzyme kinetic parameters for the probe reaction were estimated from the best fit line, using least-squares linear regression of the inverse substrate concentration versus the inverse velocity (Lineweaver-Burk plots), and the mean values were used to calculate 50IC values were 15.05, 21.83, and 28.35 μM, respectively.iK was obtained to be 7.16 μM  over the in vivo concentration of the inhibitor (Ito et al. in vivo investigations are needed for the identification of the interaction between GAA and CYPs. In addition, according to previous studies, the maximum concentration in plasma of GAA was 2.24–10.99 ng/mL after oral administration of 3000 mg Lingzhi, which is much less than the IC50 values of CYP3A4, 2D6, and 2E1. (Teekachunhatean et al. Actually, the In conclusion, this paper investigated the effect of GAA on the activity of CYPs in HLM systemically. The results showed the inhibitory effect of GAA on the activity of CYP3A4, 2D6, and 2E1, while other isoforms were not affected. These results provided direct evidence for the activity of CYPs in the presence of GAA. And it is also recommended that GAA should be used carefully with other drugs metabolized by CYP3A4, 2D6, and 2E1.


Novel aromatic aliphatic diacid monomer, 4 4ʹ bis(4-carboxy methylene) biphenyl was synthesized via friedel craft acylation with acetic anhydride followed by willgerodot reaction and used in the preparation of polyamides by direct polycondensation using the Yamazaki phosphorylation reaction with commercial aromatic diamines. These polyamides were obtained in good yield with moderate-to-high-molecular-weight build up as evidenced by inherent viscosities in the range of 0.52–0.96 dlg−1. The XRD studies showed that all the polyamides were amorphous in nature and these polymers dissolved in polar aprotic solvents such as N-methyl-2-pyrrolidone(NMP),N,N-dimethylsulfoxide(DMSO), N,N-dimethyl formamide (DMF) and N,N-dimethyl acetamide. The resulting polyamides were characterized by inherent viscocity measurements, FT-IR, 1H NMR and 13C NMR spectroscopy, solubility, thermogravimetry, X-ray diffraction studies. These polyamides had glass transition temperatures between 210°C and 261°C, and their temperatures at a 10% weight loss were 620 to 710°C and 497 to 597°C in nitrogen and air atmospheres, respectively, which indicates thermally stable polymers. However, these wholly aromatic polyamides have some drawback and limitations such as poor solubility in some common organic solvents and their extremely high melting temperatures. Most of them do not melt before decomposition because of the rigidity nature and strong intermolecular force of attraction. These properties give rise to difficulties in processing and limitations in their applications. In order to overcome these problems, flexible units such as -NHCO-, -O-, -CH2- and -SO2-, bulky pendent substituents, and non-coplanar moieties were introduced to the polymer backbone to reduce the chain interaction and rigidity nature of the polymer .In the past worked polyamides derived from biphenyl unit containing monomer by adding substituent at 2 2ʹ position on biphenyl such as methyl, cyano, phenyl, halogen, sulfonic acid and trifluoromethyl phenoxy groups . The bulWe have demonstrated an approach to make high-temperature soluble aromatic polymers with sustained thermal stability. We report the synthesis of new novel 4 4ʹ bis(4-carboxy methylene) biphenyl from biphenyl and acetic anhydride starting material via friedel craft acylation followed by willgerdot reaction, it contained methylene spacer group. The polyamides were prepared via direct Yamazaki phosphorylation reaction with commercial aromatic diamines and their properties were compared with those polymers prepared from 2 2ʹ substituent biphenyl. Prepared polyamides containing methylene spacer group in there backbone to improve the solubility, thermal properties and glass transition temperature. Methylene spacer group reduces the chain–chain interaction and intermolecular force of attraction.The structure of prepared aromatic diacid was confirmed by infrared (IR) and nuclear magnetic resonance spectroscopy (NMR) and the prepared polyamides were characterized by IR spectroscopy, inherent viscosity, solubility, thermal analysis and XRD analysis. The influence of the introduction of methylene spacer groups on the solubility and processability of aromatic polymers has been studied.We have reported polymers derived from 4 4ʹ substituent biphenyl unit with improved solubility and the retention of high thermal stability than polymers derived from 2 2ʹ substituent biphenyl unit. The attachment of methylene spacer group at 4 4ʹ position on biphenyl decreases polymer interchain interaction to reduce packing efficiency and crystallinity leads to enhanced solubility.2.2.1Acetic anhydride, anhydrous aluminum chloride, biphenyl, carbon disulphide, sulphur, morpholine. N-Methyl-2-pyrrolidone (NMP) were purified by distillation under reduced pressure over calcium hydride and stored over 4Å molecular sieves. Pyridine was refluxed over potassium hydroxide pellets, distilled and stored over 4Å molecular sieves, LiCl was dried under vacuum at 150°C for 6 hrs. Triphenyl phosphite (sigma-Aldrich). 4,4ʹ- Oxydianiline (ODA), 4,4ʹ- sulfonyl dianiline (SDA) 4,4ʹ- methylene dianiline (MDA) were recrystallized prior to use.2.1.1.The FT-IR spectra of organic compounds and polymers were recorded using Nicolet spectrometer. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were obtained with a Bruker Advance spectrometer at 25°C using CDCl3 and DMSO- d6 as solvent. Polyamides inherent viscosities were obtained with a polyamide concentration of 0.5 g/dL in DMF solvent at 30°C using an Ubbelhode suspended level viscometer. Differential scanning calorimetry (DSC) was measured on a Mettler Toledo DSC STARe instrument at heating rate of 200°C/min under nitrogen. The glass transition temperatures (Tg) were determined from DSC curves. Thermogravimetric analysis (TGA) was performed on a Mettler Toledo STARe instrument at a heating rate of 10°C/min under nitrogen. Wide angle X-ray diffraction (WAXD) was measured with a Rigaku X-ray diffractometer using polyamide powder.2.2Synthesis of 4, 4ʹ diacetyl Biphenyl: In a one litre, three-neck round bottom flask equipped with a pressure-equalizing dropping funnel, a mechanical stirrer and a reflux condenser were placed with 27.72 g (0.18 mol) biphenyl, 280 mL dry carbon disulphide and 120 gm (0.9 mol) of finely powdered anhydrous aluminium chloride. Stir the mixture and heat on oil bath until gentle refluxing commences; add 36.81 mL (0.39 mol) of redistilled acetic anhydride slowly through the dropping (30–50 minutes). The reaction mixture was stirred for 12 h at room temperature and poured in 100 ml beaker containing mixture of ice water. It was filtered and washed with cold ethanol:water mixture (70: 30). Yield 35.6 gm 84%, M. P. 192–194°C.Synthesis of 4 4ʹ bis(4-carboxy methylene) biphenyl: In a 100-mL round bottom flask equipped with magnetic stirrer and reflux condenser were placed 21.33 g (0.12 mol) 4, 4ʹ diacetyl biphenyl, 11.66 g  morpholine. The resulting reaction mixture was stirred initially under gentle reflux until the evolution of hydrogen sulphide subsided and then more vigorously for a total period of 14 h at 130°C. The reaction mixture was allowed to cool and poured into 400 mL warm ethanol and left for recrystallisation. 4, 4ʹ biphenyl thioacetomorpholide separated as pale buff crystals; it was filtered and washed with cold ethanol. Yield 35.4 g 89.77%, M. P. 230°C. The product was taken in a 1 L round bottom flask equipped with reflux condenser, magnetic stirrer to which 500 mL of 10% alcoholic NaOH were added. The reaction mixture was refluxed for 5 h, allowed to cool to room temperature and poured into 2 L water. Most of ethanol distilled out under reduced pressure. To the residual product 500 mL, hot water was added and filtered. The filtrate was acidified by 1:1 hydrochloric acid. The precipitated product was filtered, washed thoroughly with hot water and dried. The yield of 4 4ʹ bis(4-carboxy methylene) biphenyl was 15.2 g; 69.98 %, M. P. 150°C. It was recrystallized from aq. ethanol.The new aromatic diacid was synthesized in two steps; as given below.FT-IR: 2927 cm−1(-OH), 2854 cm−1 aliphatic – CH,1694.14 cm−1 (Carbonyl)1H NMR : 3.5 ppm , 3.5 , 7.2 ppm , 7.4 ppm .13C NMR : 40.93 ppm (CH2), 126, 130, 134, 138 ppm (CH),173 ppm (-COOH)2.3.A mixture of 0.270 g (1 mmol) of 4 4ʹ bis(4-carboxy methylene) biphenyl, 0.200 g (1 mmol) of diamine DA-1, 0.200 g of lithium chloride, 0.63 ml of triphenyl phosphite, 0.5 mL of pyridine, and 2.5 mL of NMP was heated with stirring at 110°C for 3 h under nitrogen atmosphere. After cooling, the reaction mixture was poured into a large amount of methanol with constant stirring, producing a fibrous precipitate that was washed thoroughly with methanol and hot water, collected on a filter, and dried at 110°C under vacuum for 24 h. The inherent viscosities in the range of 0.52 to 0.96 dlg−1. The IR spectrum (film) exhibited absorptions at 1654.45 cm−1 (C = O),2918.73 cm−1 aliphatic – CH and 3279.45 cm−1(-NH stretch of amide). All the other PAIs were prepared using a similar procedure.3.The aim of this study was to focus on improving the solubility of polyamides and thermal stability. We have been synthesized a series of aliphatic aromatic polyamides containing methylene spacer group in the main chain of polymer backbone by Yamazaki’s phosphorylation method to improve the solubility and inherent viscosity of these polyamides. These aliphatic aromatic polyamides showed inherent viscosities in the range of 0.52–0.96 dl g−1 as shown in 3.1The 4 4ʹ bis(4-carboxy methylene) biphenyl monomer was synthesized in two steps (Scheme 1). In the first step, 4 4ʹ diacetyl biphenyl was prepared from biphenyl and acetic anhydride in the presence of lewis acid catalyst anhydrous aluminum chloride, which undergoes fridal craft acylation reaction on biphenyl. In the second step, 4 4ʹ bis(4-carboxy methylene) biphenyl was prepared from willgerdot reaction of 4 4ʹ diacetyl biphenyl (scheme 1).3.2The chemical structure of 4 4ʹ bis(4-carboxy methylene) biphenyl monomer, 4 4ʹ bis(4-carboxy methylene) biphenyl was confirmed by FT-IR and NMR (1H 13C). FT-IR spectrum of 4 4ʹ bis(4-carboxy methylene) biphenyl showed two characteristic stretching absorption band at 2927.27 cm−1 due to the hydroxyl group (–OH) of carboxylic acid, 2854 cm−1 due to the aliphatic – CH stretching and absorption band at 1694 cm−1 due to the carbonyl stretching as shown in 3.3The aliphatic aromatic polyamides were prepared by direct polycondensation of 4 4ʹ bis (4-carboxy methylene) biphenyl with various aromatic diamines in NMP by the Yamazaki et al. method using TPP and Pyridine as condensing agents (scheme 2). The obtained polyamides were quantitative yields with inherent viscosity in the range of 0.52–0.96 dlg−1. All structural features of these resulting polyamides were verified by FT-IR . They ex3.4The solubility was qualitatively determined by the dissolution of 1 mg of polymer sample in 9 mg of solvent (10 wt%) at room temperature or an elevated temperature, and the results are summarized in The solubility studies also revealed that solubility of polyamides enhances due to the incorporation of methylene spacer group at 4 4ʹ position on biphenyl as comparably to the spacer group present at 2 2ʹ position on biphenyl in which 4 4ʹ substituent at biphenyl decreases interchain interaction and increase free volume in polymer backbone than that of 2, 2ʹ substituent at biphenyl .The X-ray studies revealed that all the polymers were completely amorphous in nature. Thus, the amorphous nature of these polymers was reflected in their excellent solubility and this could be attributed to the incorporation of methylene spacer group at 4, 4ʹ position on biphenyl unit along the polymer backbone.3.5The thermal behavior and stability of the prepared polyamides were evaluated by means of TGA and DSC, and the results are summarized in Figure 6Structure-thermal property correlations based on the obtained carbonized residue (char yield) led to several interesting marks:The properties of the polyamides depend not only on the structural composition but also on regularity of structure. All the polyamides begin to decompose at about 229°C. The TGA of polyamides (PA-I to PA- V) shows T10% weight loss temperature of the polyamides at 588 °C (PA-I), 532°C (PA-II), 507°C (PA-III), 564°C (PA-IV), 499°C (PA-V), and the amount of carbonized residue (char yield) value was 46.13% (PA-I), 59.90% (PA-II), 56.17% (PA-III), 62.14%(PA-IV), and 61.40% (PA-V) at 800 °C, respectively, as shown in The polyamides PA-III and PA-V contain sulfone linkages and heterocyclic thiophene unit in the polymer backbone, respectively. These polyamides show lower T10% weight loss residue temperature than the other polyamides. This might be attributed to the nature of weak bond between carbon and sulphur (C-S) and easy degradation in heating process.The amount of carbonized residue (char yield) value at 800°C of polyamides containing pyridine (PA-IV) and thiophene (PA-V) moiety are more than the other polyamides containing spacer methylene (PA-II), sulfone linkages (PA-IV) and flexible ether (PA-I) moiety, due to the rigid or stiffness nature of heterocyclic ring. A higher order of thermal stability is achieved in these polyamides. Strong intermolecular binding and chain stiffness are also contributing factors for these polyamides.Overall, the heterocyclic ring containing polymers exhibited better thermal stability than their analogues containing bridging sulfone, spacer methylene and flexible ether groups or moiety.Comparative Thermal Stability Study of Polyamides containing Biphenyl Unit (PA-1) with other Biphenyl unit having Flexible, Spacer and Pendant groupsOn comparing as shown in the above figure, the thermal stability of polyamides  with respect to T10% of the structurally related polyamides containing biphenyl unit with Pendant Flexible, Spacer groups in the polymer chains.The order of thermal stability of biphenyl is as follows: The PA-01  polyamides (588°C) > Two -CF3 and Binaphthyl unit containing polyamides (549°C) > polyamides based on Pendant Phenyl Group (polyamide[> Two Pendant Methyl group cantered Polyamides (494°C) [30] > Two -CF3 Group containing Polyamides (490°C) > Two -CF3 and four pendant Methyl group covered Polyamides .From the above thermal data study, we have concluded as the insertion of the Two -CF3 group and increase in the number of Pendant Methyl group reduces the thermal stability of Polyamides . But if 4.A new 4 4ʹ bis(4-carboxy methylene) biphenyl monomer was successfully synthesized via fridal craft acylation reaction and willegrdot reaction with good yield. Aliphatic aromatic polyamides were synthesized from 4 4ʹ bis(4-carboxy methylene) biphenyl monomer with various aromatic diamines by Yamazaki phosphorylation reaction. Prepared polymers appeared amorphous in nature with good solubility in many aprotic solvents. Incorporation of methylene spacer group in polymer backbone increases the solubility and thermal stability of polymers.From the above result, it was confirmed that the introduction of the methylene spacer group at 4 4ʹ position on biphenyl into the polymer backbone was highly effective for the preparation of soluble aromatic polyamides having both high Tg and high T10 value than the polymers derived from 2 2ʹ substituent biphenyl unit. The polymer we have developed could be used as a high-performance polymeric material.


The combination of MYB and bHLH in MBW is responsible for a wide range of biological events, while the variation of WD-repeat protein is low. In Arabidopsis thaliana, GL3, bHLH, forms a complex with GL1, MYB, and the GL3-GL1 complex generates a trichome in epidermal cells, which is believed to protect the plant body from insects. GL3 also forms a complex with PAP1, another MYB, and the GL3-PAP1 complex regulates the synthesis of anthocyanin which protects against UV and strong light . Because MBW consists of various MYBs, a competition between MYB to bHLH impacts the activity and the regulation of gene expression . Thus, the affinity between MYB and bHLH proteins are essential for understanding the mechanism underlying the combinatorial regulation of MBW complex . However, little is known about the quantitative evaluation of affinities between MYBs and bHLH. To acquire affinity information of MBW in a quantitative manner, we focused on GL3, GL1, and PAP1. Because these proteins are expressed in an epidermal cell at early leaf development, GL3, GL1, and PAP1 would integrally impact the trichome formation and anthocyanin biosynthesis via competitive interactions. We quantified the dissociation constants of these three proteins using a quartz crystal microbalance (QCM). QCM is a biosensor to monitor in real time an interaction that occurred on a sensor chip by a frequency of a quartz crystal resonator  . We purified the recombinant proteins of GST-tagged GL3, His-tagged GL1, and GST-tagged PAP1, which were expressed in bacteria and confirmed using SDS electrophoresis . Purified GL1 and GL3 were separately immobilized on the surface of two sensor chips. The immobilization was observed as a difference of frequency. Following the confirmation of stable immobilization, we proportionally added the other proteins, GL3 or PAP1, in a cuvette, because the difference of frequency represents the amount of correlated proteins. Dissociation constants were measured by applying nonlinear fit on Langmuir formula , resulting in the dissociation constants (DK) of GL3 and GL1, GL3 and PAP1, and PAP1 and GL1, which were 9.53 ± 7.52 µM, 0.29 ± 0.20 µM, and 0.049 ± 0.04 µM, respectively (average of three independent experiments) . Previously, the DK values between EGL3, GL3, and PAP2 were described using a different system from ours, wherein the range of DK between bHLH and MYBs was 3.5–7.5 µM . Since we can not rule out that the GL3-PAP1 interaction is due to GST dimerization, our conclusion should be considered as preliminary. Nevertheless, these DK values are in a similar range with the GL3-GL1 interaction in our study. It is notable that our result shows that the affinity between GL1 and PAP1 was 100-fold higher than that between GL3 and GL1 . These results propose that the competitive regulation in the MBW complex should be considered not only between bHLH and MYB but also between MYBs.Plants protect themselves from different environmental stresses, and the responses are governed by multiple transcription factors (TFs) via complex forms. Plants evolutionally conserve a heteromeric complex, comprised of MYB, basic helix-loop-helix (bHLH), and WD-repeat proteins (MBW) (Albert Construction of expression vectorsCDS regions of GL1 (At3g27920), GL3 (At5g41315), and PAP1 (At1g56650) were amplified by PCR with Prime STAR  from the pDONAR vectors containing the GL1, GL3, and PAP1 CDS provided by Nobutaka Mitsuda  with following oligo DNA primers, respectively. MU_GL1_F, 5’-AATGGGTCGCGGATCCGAAATGAGAATAAGGAGAAGAGATG-3’; MU_GL1_R, 5’-TGTCGACGGAGCTCGAATTAAGGCAGTACTCAACATCACC-3’; MU_GL3_F, 5’-GCCCCTGGGATCCCCGGAAATGGCTACCGGACAAAACAGAA-3’; MU_GL3_R, 5’-TCGAGTCGACCCGGGAATTTCCTGATGATGATGACGATGA-3’; MU_PAP1_F, 5’-GCCCCTGGGATCCCCGGAAATGGAGGGTTCGTCCAAAGGG-3’; MUPAP1_R, 5’-TCGAGTCGACCCGGGAATTATCAAATTTCACAGTCTCTCC-3’. The DNA fragment was cleaved by EcoR1 and was cloned into the Shrimp Alkaline phosphatase treated pGEX6P-1 vector using HiFi DNA Assembly Master Mix . For GL1, construction was also created in the pET21a-d vector; these constructs were introduced into Rozetta 2 (DE3) by the heat shock transformationPurification of recombinant proteinsSingle colonies of Rozetta 2 (DE3) transformed with GL1-pGEX6P-1, GL3-pGEX6P-1, PAP1-pGEX6P-1, and GL1-pET21a-d were grown in 50 mg/L carbenicillin containing LB liquid medium at 37˚C. 3 ml of pre-cultured cells was added to 300 ml of LB liquid medium containing 50 mg/L carbenicillin and cultured at 37°C until the OD600 reached 0.6. IPTG  was added at a final concentration of 1 mM, and induction was carried out at 20°C for 20 hours. After induction, the cells were collected for 15 minutes at 3500 rpm and washed once with 50 ml of PBS. In the case of GST and GST-fused proteins, cells were suspended in 30 ml of ice-cold GST buffer ((20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% TritonX, 1 mM PMSF, 5 mM DTT, 1 mM PMSF, 1 mM protease inhibitor cocktail), 3 ml of 10 mg/ml lysozyme was added and incubated for 30 minutes at 37° C. Sonication was performed for 30 seconds for four times on ice. 300 μl of Triton X-100 was added, and the mixture was stirred at 4° C for 15 minutes by a rotator, followed by centrifugation at 9,000 rpm for 15 min at 4°C. 1 mL of Glutathione-Sepharose 4B(GE), which washed twice with ice-cold GST buffer, was added to the supernatant, and the mixture was stirred at 4°C overnight by a rotator. After centrifugation at 5,000 rpm for 5 min at 4°C, the supernatant was removed, and the beads were washed three times with 10 ml of ice-cold GST buffer (1 mM DTT), and proteins were eluted by 2.0 ml of High Salt Elution Buffer , 120 mM NaCl). In the case of His-GL1, precipitated induced cells were resuspended to 30 ml of ice-cold TKET buffer  containing a final concentration of 10 mM Imidazole. 3 ml of 10 mg/ml lysozyme was added, and the mixture was incubated at 37°C for 30 minutes. The mixture was sonicated for 30 seconds for four times on ice. 300 ul of Triton X-100 was added, and the mixture was stirred at 4° C for 15 minutes by a rotator. After centrifugation at 9,000 rpm for 15 min at 4°C, 1 ml of Ni-sepharose 6FF(GE), which was washed twice with ice-cold TKET buffer (10 mM Imidazole), was added to the supernatant, and the mixture was stirred at 4°C overnight by a rotator. After centrifugation at 5,000 rpm for 5 min at 4°C, the supernatant was removed, and the beads were washed three times with 10 ml of ice-cold TKET buffer (10 mM Imidazole). Proteins were eluted with ice-cold TKET buffer (300 mM Imidazole).Protein sample preparation10 ul SDS sample buffer (125 mM 0.5 M Tris-HCl buffer (pH 6.8), 4% SDS, 20% glycerol, 0.01% BPB, 10% 2-mercaptoethanol) was added to 10 ul of purified protein. Samples were boiled for 5 minutes at 90°C. Then, SDS-PAGE was performed by a 10% acrylamide gel, followed by CBB staining. After confirmation of a single band, the buffer of purified proteins was exchanged with PBS  using Amicon Ultra . The protein concentration was measured with Pierce BCA Protein Assay Kit , and ultrafiltration was performed until the concentration reached 750 ug/ml or more.Dissociation constant measurementImmobilization was performed according to the manufactural protocol of the immobilization Affinix kit . Shortly, the sensor chip was initialized. The electrode was carefully rubbed with a cotton swab soaked in 1% SDS, washed with running DW water, and dried by blowing air. Next, 2 μl of piranha solution (concentrated sulfuric acid: 30% H2O2=3:1) prepared in a glass container was added only on the gold film of the sensor chip, and the mixture was allowed to stand for 5 minutes. It was rewashed with running water of DW and dried by blowing air. This step was performed twice in total. An appropriate attached buffer was selected and adjusted to 100 ul . After stuffing the edges of the plastic petri dish with moistened paper and arranging the ceramics sensors, 50 μl of the SAM solution was mounted on the sensor gold film, shielded with aluminum foil, and allowed to stand at room temperature for 1 hour. After drying by blowing air, 50 μl of a solution obtained by mixing equal ratio of NHS solution and EDC solution was mounted on the gold film, shielded from light, and allowed to stand at room temperature for 15 minutes. After washing with DW running water and air, the frequency of quartz was measured. 100 µl of the prepared protein solution was mounted on the gold film. After shielding from light and standing at room temperature for 1 hour, the air was blown to dry it, and then the frequency of quartz was measured again. Washing and air were repeated until the frequency of quartz was lower than the measured value by 1000 Hz. 100 μl of ethanolamine was mounted, and protected from light, and allowed to stand at room temperature for 10 minutes. A frequency was measured again and used as the final fixed amount.The protein-immobilized sensor was attached to Affinix Q  and placed in a glass cell containing 8 ml of PBS to stabilize it. After the output of the frequency of quartz become stable, 1 to 32 ul of the protein solution was injected. Protein solutions with different volumes were injected multiple times. Then, the dissociation constant was calculated by AQUA . The measurement conditions were set at a set temperature of 25°C, a stirrer rotation speed of 1,000 rpm, and a data acquisition speed was 1 second. The experiments were repeated at least three times by using different sensor chips in each measurement of interaction.


CRHs were used as additives to improve the thermal conductivities of the PCMs. At lower fixed temperatures such as 30 to 32 °C and 35 to 37 °C, the rates of increase of the surface temperatures of roofs containing CRHs with Na2SO4·10H2O, and paraffin wax, were observed to gradually decrease compared to those of the roofs without PCMs. The indoor temperatures for the above-mentioned PCMs containing CRHs were maintained to be lower than those of the indoors without PCMs. Additionally, as the CRH content in the PCM increased, the rates of increase of the roof-surface and indoor temperatures decreased due to a faster roof heat absorption by PCMs through the improved thermal conductivity of CRHs. However, under higher artificial temperatures such as 40 to 42 °C, Na2SO4·10H2O with CRHs exhibited no effect due to being out of latent heat range of Na2SO4·H2O. For paraffin wax, as CRH content increased, their roof- surface and indoor temperatures decreased. Especially, the surface temperature of the roof containing paraffin contained 5 wt.% CRHs reduced by 11 °C, and its indoor temperature dropped to 26.4 °C. The thermal conductivity of PCM was enhanced by the addition of CRHs. A suitable PCM selection in each location can result in the reduction of the roof-surface and indoor temperatures. This study researches the effect of phase change materials (PCMs) containing carbonized rice husks (CRHs) in wood plastic composites (WPCs) as roof finishing materials on roof-surface and indoor temperatures. A cool roof miniature model was prepared, and measurements were taken using three fixed temperatures of 30 to 32 °C, 35 to 37 °C, and 40 to 42 °C. Sodium sulfate decahydrate (Na Direct exposure of construction surfaces to sunlight has been observed to increase the temperature in buildings; this is because of the higher heat storage and lower heat emission of materials such as concrete or metal deck ,3,4,5,6.PCMs used for cool roof system applications are materials capable of absorbing or releasing thermal energy in the form of latent heat during the solid–liquid transition, and are extensively applied in many energy economy areas due to sustained heat source temperature, high energy density, and repeated utilization ,18,19,20Nevertheless, PCMs have major disadvantages such as supercooling and phase segregation due to low thermal conductivity, corrosion, and latent heat loss during liquid–solid repetition. Therefore, to inhibit these disadvantages, various additives have to be added to PCMs. Carbonized materials are widely used to improve the thermal conductivity, which results in the prevention of supercooling and phase segregation. Additionally, they possess favorable properties such as noncorrosion by contact with PCMs, chemical stability, nontoxicity, and being lightweight ,34,35,36The aim of this study was to research the attainment of reduced roof-surface and indoor temperatures through fast roof heat absorption by improving the thermal conductivity of PCMs through the addition of CRHs. In addition, the study aimed to minimize the energy loss in latent heat storage and retrieval progress by removing the phase segregation that is not completely soluble during melting and the supercooling problems that remain molten PCMs during crystallization to ensure timely release of the heat of fusion. To apply the cool roof system, a cool roof miniature model was prepared. 2SO4·10 H2O (phase change temperature of 32.4 °C with a purity greater than 98.0%) was received from DAEJUNG Co. , and Paraffin wax  was supplied from WR Medical Electronics Co. . Wood plastic composite (WPC) structure samples were obtained from YES WOOD Co. in Korea.Rice husks were purchased from SAMWHA RICE MILL Co. in Korea. PCMs were used as received; Na2 (99.999%) without a stabilization procedure. The obtained CRHs were used as additives to improve the thermal conductivity of PCMs.The CRHs were carbonized for 1 h at a temperature of 1000 °C in muffle furnace under an atmosphere of N2SO4·10H2O/CRHs and paraffin wax/CRHs in PE bags. The density increased with the increase of CRHs. 2SO4·10H2O/CRHs and paraffin wax/CRHs obtained during cooling from the melt in the PE bags. It can be observed that the CRHs were impregnated within the Na2SO4·10H2O and paraffin wax were uniformly dispersed. PCM/CRHs (100 g) packs were prepared by physically mixing the PCM powders evenly with 0, 1, 3, 5 wt.% CRHs, respectively, and then inserting the mixture in polyethylene (PE) bags. Hot pressing the ends of the PE bag enclosed the bags. The end of PE bag containing PCM/CRH were closed using a hot press. 3) and cooled at room temperature to yield PCM/CRH composites that were used to measure the thermal conductivity. The latent heat of the PCM/CRHs was measured using differential scanning calorimetry  at a heating and cooling rate of 1 °C min−1 in a nitrogen atmosphere. The samples were prepared by cooling at room temperature after mixing the melted PCM with 0, 1, 3, 5 wt.% CRHs. Approximately 10 mg of the samples was used for the DSC analysis. Estimation was performed based on the respective endothermic areas of the DSC curves during PCM melting with TA Instruments TRIOS v4.4.0 software package. X-ray diffraction (XRD) pattern was recorded by a RIGAKU, D/MAX-2500 instrument  with CuKα radiation generated at 40 kV and 30 mA at a scan rate of 0.4 °/min. The thermal conductivity was measured using a TPS2500S instrument ; the data was acquired using a sensor sandwiched between two identical PCM/CRH composites. The PCM/CRHs composites used for the thermal conductivity measurements were obtained through a melting method. The PCMs were melted at their respective phase change temperatures followed by the addition of 0, 1, 3, 5 wt.% CRHs. The resulting mixtures were placed in a cuboid shape bowls ,  and (100) planes. The (002) peak results from the graphitization conversion, and thus demonstrated the successful carbonization of the rice husks. 2SO4·10H2O and paraffin wax increased with increased CRH content due to the higher contact and more dense per-unit area of CRHs, and the thermal conductivities of pure Na2SO4·10H2O were higher than those of pure paraffin wax. Therefore, thermal conductivities of Na2SO4·10H2O/CRHs are generally higher than those of paraffin wax/CRHs under similar conditions.2SO4·10H2O was 216.13 ± 2.70 kJ kg−1 at the melting temperature of Tm of 32.4 °C, and paraffin wax was 220.45 ± 1.62 kJ kg−1 at Tm of 48 °C. Generally, PCMs presented one endothermic peak at melting temperature; however, in the case of paraffin wax, double endothermic peaks were observed. The first peak represents the solid–solid conversion, which indicates the change of the ordered phase into the disordered phase, and the second peak, which is the main peak, indicates the solid–liquid conversion. Therefore, in 2SO4·10H2O (27~41.5 °C). The latent heat values of PCMs gradually decreased as the CRH content was increased in 2SO4·10H2O and paraffin wax increased with an increase of CRHs. In 2SO4·10H2O was observed at approximately 0 °C; however, Na2SO4·10H2O-containing CRHs crystalized at a higher temperature of approximately 10 °C. This is because the thermal conductivity of Na2SO4·10H2O is more improved with the increase of CRHs. In addition, as shown in m and at a slightly higher temperature with an increase in CRHs. These results indicate that the addition of CRHs, especially in the case of Na2SO4·10H2O, might prevent the supercooling and phase segregation difficulties by improving the thermal conductivity of PCMs.2SO4·10H2O/CRHs using the different fixed temperatures, on the roof-surface and the indoor temperatures. As depicted in 2SO4·10H2O or Na2SO4·10H2O/CRHs. These temperatures in 2SO4·10H2O-containing CRHs showed a lower increase than that for roof with only Na2SO4·10H2O. Indeed, as the CRH content increased, the roof-surface temperature increased at a slower pace and resulted in a lower overall temperature, compared to the roof with no PCM, and the indoor temperature showed a decreasing trend. These results are due to a faster roof heat absorption through the improved thermal conductivity of CRHs; this led to a faster melting of Na2SO4·10H2O. However, at the fixed temperature of 40 to 42 °C in 2SO4·H2O comprising 5 wt.% CRHs was observed to have only a slight difference of approximately 1°C, compared to the roof without Na2SO4·10H2O. This is due to it being out of range of the Na2SO4·10H2O latent heat (see 2SO4·10H2O or Na2SO4·10H2O with CRHs were used at the fixed temperature of 40 to 42 °C; however, the indoor temperatures were observed to keep lower for roofs containing Na2SO4·10H2O or Na2SO4·10H2O with CRHs than those of roof without PCM.heat see a. Theref2SO4·10H2O with CRHs. In the cases of the fixed temperatures of 30 to 32 °C and 35 to 37 °C, when compared to roofs without PCM, the roof-surface temperatures with paraffin wax with CRHs and without PCMs showed a maximum difference of approximately 7 °C and 11 °C, respectively; further, the indoor temperatures exhibited a maximum difference approximately 4 °C and 6 °C, respectively. However, at the fixed temperature of 40 to 42 °C, as the CRH content increased, the roof-surface temperatures decreased. In particular, the surface temperature of roofs containing paraffin wax with 5 wt.% CRHs reduced to 74.6 °C; further, the indoor temperature also decreased to 26.4 °C, even though the roof-surface and indoor temperatures without PCM reached 86 °C and 35.5 °C, respectively. Therefore, it has been concluded that selection of PCMs with higher Tm than the summer temperatures of each location can significantly influence the reduction of roof-surface temperatures and indoor temperature in buildings.2SO4·10H2O and paraffin wax with CRHs increased at a slower rate than those of the roofs without PCMs. The indoor temperatures of the roofs containing Na2SO4·10H2O and paraffin wax with CRHs were also maintained to be lower than those of the roofs without PCMs. Additionally, as the CRH content in the PCM increased, the rate of increase of the roof-surface and indoor temperatures also decreased because of a faster heat absorption of the roof surfaces by PCMs due to the improved thermal conductivity through CRHs. However, under a fixed temperature of 40 to 42 °C, the surface temperature of the roof containing Na2SO4·10H2O with 5 wt.% CRHs increased to approximately 80 °C, which was similar to that of the roof without PCM. This was due to being out of range of the latent heat of Na2SO4·10H2O; however, the indoor temperatures containing Na2SO4·10H2O with CRHs were still maintained to be lower than those of the roofs without PCMs. In the case of paraffin wax with Tm of 48 °C and under the fixed temperature of 40 to 42 °C, the CRH content was observed to increase, and the roof-surface temperatures decreased. In particular, when compared to roofs without PCMs, the surface temperature of roof containing paraffin wax with 5 wt.% CRHs reduced to 74.6 °C of a maximum difference of approximately 11 °C and its indoor temperature was also lowered to 26.4 °C. In this study, the improvement of the thermal conductivity of PCM by the addition of the CRHs and a suitable PCM selection in each location can result in the reduction of the roof-surface and indoor temperatures.This study aimed to obtain lower roof-surface and indoor temperatures through the fast heat absorption of PCMs in WPC of roof finishing materials; this was achieved by improving the thermal conductivity of PCMs through CRHs. Under the fixed temperature of 30 to 32 °C and 35 to 37 °C, the surface temperature of the roofs containing Na