Tlxyn11B, in Talaromyces leycettanus JCM12802. Recombinant TlXyn11B produced in Pichia pastoris is distinguished by high specific activity (8259 ± 32 U/mg with beechwood xylan as substrate) and excellent pH stability (from 1.0 to 10.5). The beechwood xylan hydrolysates consisted mainly of xylobiose, xylotriose and xylotetraose, thus TlXyn11B could be used for the production of prebiotic xylooligosaccharide. By using the structure-based rational approach, the N-terminal sequence of TlXyn11B was modified for thermostability improvement. Mutants S3F and S3F/D35V/I/Q/M had elevated Tm values of 60.01 to 67.84 °C, with S3F/D35I the greatest. Homology modeling and molecular dynamics (MD) simulation analysis revealed that the substituted F3 and I35 formed a sandwich structure with S45 and T47, which may enhance the overall structure rigidity with lowered RMSD values. This study verifies the efficiency of rational approach in thermostability improvement and provides a xylanase candidate of GH11 with great commercialization potential.Thermophilic xylanases with high catalytic efficiency are of great interest in the biofuel, food and feed industries. This study identified a GH11 xylanase gene,  L-arabinofuranosidase (EC 3.2.1.55), acetyl esterase (EC 3.1.1.72), ferulic acid esterase (EC 3.1.1.73), and p-coumaric acid esterase (EC 3.1.1.x)1. Endo-1,4-β-xylanase as the key enzyme decomposes inherent heteroxylans into short oligosaccharides or (and) xylose by randomly attacking the β-1,4-linkage in the xylan backbone2, and assists cellulase to access cellulose.Xylan accounts for approximately 35% of the dry weight of plant cell wall and represents one of the most important, abundant, and renewable bioresources. It is cross-linked with lignin and cellulose, which hinders it from efficient utilization. Due to the complex structure, its complete degradation requires the synergistic action of xylanolytic enzymes including endo-1,4-β-xylanase (EC 3.2.1.8), 1,4-β-xylosidase (EC 3.2.1.37), α-glucosiduronase (EC 3.2.1.139), α-3. According to the sequence identity and three-dimensional structure homology, xylanases are classified into glycoside hydrolase (GH) families 5, 8, 10, 11 and 30 (http://www.cazy.org/), with the majority in GH10 and 114. GH10 xylanases have typical triosephosphate isomerase (TIM) structures with greater molecular mass, while those from GH11 share a right hand β-jelly roll structure and are industrially attractive due to the small size, high substrate selectivity, and favorable pH and temperature optima3.Xylanases have been widely applied in many fields, such as pharmacy, food and feed industry, biofule, textile, etc6. The directed evolution is a robust strategy to engineer enzymes by accelerating protein evolution7. However, this method involves in construction of large libraries and laborious screening. In contrast, the rational design derived from previous studies and sequence analysis is less laborious and time consuming8. Recently, computer-aided approaches have been used to analyze the primary sequence, simulate the tertiary structure and de novo predict the protein interaction9. Based on these bioinformatic analyses, key amino acids are identified and site-directed mutagenesis is conducted. This in silico rational method thus dramatically reduces the library size and workload and increases the engineering efficiency as well, and is widely used for protein improvement.To meet the industrial demands, two protein engineering approaches, i.e. directed evolution and rational design, are commonly used to improve enzyme properties12. However, their wide applications have been limited by the weak thermostability. It is found that the N-terminal sequence plays a key role in the stability of GH11 xylanases. By replacing the N-terminus with thermostable ones14, introducing a disulfide bond to the N-terminus15, substituting the N-terminal residues16, and optimizing the surface charge interactions17, the thermostability of GH11 xylanases have been successfully improved. The underlying mechanism has been ascribed to the more amino acid interactions and decreased unfolding entropy13. In this study, a highly active GH11 xylanase with a broad pH adaptability range but weak thermostability was identified in Talaromyces leycettanus JCM12802. To improve its thermostability, a rational design focused on the N-terminal region was conducted. As results, a mutant with greater thermostability was obtained, and the underlying mechanism was then revealed by the analysis of molecular dynamics simulation.A large number of GH11 xylanases have been cloned from fungi and bacteria and biochemically characterizedTlxyn11B (GenBank accession No. KY594261) contains 654 bp that encodes a 217 amino acid-residue polypeptide. The N-terminal 19 amino acid residues were predicted to be a putative signal peptide, and the mature protein was predicted to have a calculated molecular mass of 21.9 kDa and a pI of 4.63. The deduced amino acid sequence of TlXyn11B shares the highest identity of 84% with the xylanase A of Penicillium sp. 40, and of 73% with the structure-resolved GH11 endo-1,4-β-xylanase from Talaromyces cellulolyticus (PDB: 3WP3). By using the crystal structure 3WP3 as template, modeled TlXyn11B showed a β-jelly roll structure of typical GH11 xylanases and an extended N-terminus at the bottom of the palm region  with steepest decent methods and conjugate gradient methods indicated that the conformation of this N-terminus has no tremendous changes before and after energy minimization. Thus we conjectured that certain interactions occurred in the N-terminus of TlXyn11B, which probably contribute to the fixation of this local structure at the bottom of the palm region.The full-length cDNA of TlXyn11B without the signal peptide sequence was successfully expressed in P. pastoris GS115. The culture supernatants of the positive transformant with highest xylanase activity were then purified to electrophoretic homogeneity as shown in Supplementary Fig. N-glycosylation site of TlXyn11B might link glycans during heterologous expression in P. pastoris. With and without the Endo H treatment to remove N-linked glycans, the purified enzymes migrated two single bands of almost similar apparent molecular weights (approximately 22 kDa), which are in agreement with the calculated value of TlXyn11B (21.9 kDa). This result indicated that no or little N-glycosylation occurred in TlXyn11B. Further mass (MS) analysis verified the identity of the recombinant proteins as TlXyn11B, since the polypeptide sequences of the target band were consistent with the sequence of deduced TlXyn11B   10 mM. Using beechwood xylan as the substrate, the specific activity, Vmax, Km, and kcat/Km of TlXyn11B were determined to be 8,300 ± 32 U/mg, 12,800 ± 42 U/mg, 3.3 ± 0.6 mg/mL, and 1,420 ± 26 mL/s/mg, respectively..0) Fig. , but ret.0) Fig. . DiffereTlXyn11B consisted mainly of xylobiose (25%), xylotriose (42%) and xylotetrose (29%)  Fig. . This re19, while the latter located on the bottom of a palm region tends to expose in the solution and interact with the N-terminus. Saturated mutation was conducted at residues S3 and D35 sequentially. The first round screening indicated that mutant S3F had enhanced thermostability. Based on this, saturated mutation at D35 in combination with S3F was performed. And four double mutants, i.e. S3F/D35V, S3F/D35Q, S3F/D35I and S3F/D35M, showed improved thermostability.S3 and D35 were selected for mutation due to their high B-factor values and special positions. The former is located at the N-terminus, which has been verified to affect thermostabilityTm of wild type was 60.0 °C, which was 2.3, 4.7, 5.8, 6.6 and 7.8 °C lower than those of mutant S3F, S3F/D35Q, S3F/D35M, S3F/D35V and S3F/D35I, respectively.The catalytic and thermal properties of the wild type and mutant enzymes S3F and S3F/D35V/Q/I/M were then compared. Interestingly, all mutants showed improved thermostability without activity reduction. With beechwood xylan as the substrate, all mutants had specific activities comparable to or greater than the wild type Table . In termTlXyn11B, S3F and S3F/D35I were calculated with the first conformations as references, and mutant S3F/D35I showed a rather stable profile with lowered RMSD values  values of ues Fig. . Structu35I Fig. –d. NamelTlXyn11B, was identified in T. leycettanus JCM12802. It was acidophilic and thermophilic, showing maximal activity at pH 3.5 and 65 °C and great stability from pH 1.0 to 10.5. TlXyn11B had similar pH and temperature optima to the GH11 xylanases from Talaromyces versatilis20 and Penicillium occitanis21, but retained more activity at pH 2.0 and lost activity at pH 6.0 and above. These acidic xylanases are entirely different from neutral and alkaline xylanases of GH11  in functional pH25. Although the temperature optimum (65 °C) is similar to many xylanase counterparts, its thermostability is much worse than the thermostable Thxyn11A24 and Xyn11A26. Moreover, the pH stability range of TlXyn11B is much broader than those of XYNF11a23, XynA27 and XynC8328. TlXyn11B had a specific activity of 8,400 U/mg, which is much higher than most GH11 xylanases29. In addition, its hydrolysis products were mainly xylooligosaccharides, thus TlXyn11B has potential to generate prebiotic xylooligosaccharides for feed and food purposes31.In this study, a highly active xylanase, 32. For example, by introducing a disulfide bond, the Tm value of TLX was elevated from 66 °C to 74 °C15, and the XYNII from Trichoderma reesei had increased thermostability by 11 °C33. It means that the covalent interaction between the N-terminal sequence and the palm region is significant in enhancing thermostability. Therefore, a rational design of the N-terminal sequence was carried out to engineer TlXyn11B in the present study. After two rounds of site-saturated mutagenesis, double mutation S3F/D35I contributed to the enhanced ∆Tm value of 7.8 °C and improved the thermostability of TlXyn11B at 50–70 °C. The Tm value of the surface charge engineered XynA17 was improved from 330.4 K to 332.4 K, which was significantly lower than the improvement of TlXyn11B (333.0 to 340.8 K); on the other hand, the TlXyn11B activity  was much higher than the mutated XynA activity (47 U/mg). Another intriguing finding is that the improved thermostability has no impairment on the enzyme activity  using the RING web server provides some important information  and Val (0.5%) are seldom. Similar situation occurs to Ser3, which only accounts for 5% frequency (higher than the 1% of Phe). It suggests that majority of GH11 xylanases are probably thermolabile since those thermo-related amino acids only exist in minor groups. The introduction of amino acids with low frequency (i.e. S3F and D35I) may introduce more noncovalent interactions (such as Ser45-Phe3-Thr47/Ile35) to fix the N-terminus at the protein bottom and make the whole protein structure more rigid. Therefore, this computational rational design approach is suitable for engineering local conformation with sequence diversity, such as the N-terminus and loops, in which the mutated residues fit well with adjacent amino acids.15. As both Phe3 and Ile35 exist in GH11 xylanases at an extremely low frequency, Ser45-Phe3-Thr47/Ile35 may be a new conformation in the N-terminal stabilization. Structure search using the Dali server indicated that there was no homolog structure of the N-terminus of mutant S3F/D35I in the PDB database.These introduced noncovalent interactions may cause similar effects to the disulfide bond introduced in TLXIn conclusion, a highly active GH11 xylanase with broad pH stability range was identified, produced and characterized. Protein engineering of two N-terminal residues by saturated mutation was successful to produce four double mutants with improved thermostability and no activity loss. This study not only provides an excellent xylanase candidate for xyloologosaccharide production, but also verifies the efficiency of rational design approach in engineering the N-terminus for thermostability improvement.T. leycettanus JCM12802 was grown in potato dextrose Broth (PDB). Escherichia coli Trans1-T1 was used for gene cloning. Vector pPIC9  was used for gene expression by being transformed into Pichia pastoris GS115. Beechwood xylan was purchased from Sigma-Aldrich . The DNA purification kit, LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa . The total RNA isolation system kit and T4 DNA ligase were purchased from Promega . All chemicals were of analytical grade and commercially available.Tlxyn11B, was identified in the genome of T. leycettanus JCM12802 (the whole genome sequencing in progress). After three days’ growth in induced medium, the total RNA was extracted from the mycelia and purified using the Promega SV Total RNA Isolation System according to the manufacturer’s instructions. The total RNA was used for cDNAs synthesis in vitro using the ReverTra Ace-a-™ kit . The cDNAs was used as a template for PCR amplification with specific primers  was used to identify the cDNA sequence of TlXyn11B. Clustal Omega  was used to align the nucleotide and amino acid sequences, and the results were demonstrated by ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Proparam tool (http://web.expasy.org/protparam/) was used to predict the molecular mass and theoretical isoelectric point (pI). SignalP4.0 server  was used to predict the signal peptide. Modeller 9.13 was used to build the homology model of TlXyn11B with Chimera 1.10 (http://www.cgl.ucsf.edu/chimera/) to process energy minimization. NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/) was applied to predict the potential N-glycosylation site. Pymol 0.99rc was employed to represent the homology model and aligned structures. The catalytic amino acid residues, amino acids frequency and high β-factor sites were predicted by Hotspot Wizard34 (http://loschmidt.chemi.muni.cz/hotspotwizard/). Amino acid interactions were predicted by Residue Interaction Network Generator (RING) web server35 (http://protein.bio.unipd.it/ring). Dali server  was used to search similar structures in the PDB database.BLASTx program  at 4 °C overnight and loaded onto a HiTrap Q Sepharose XL 5 mL FPLC column  equilibrated with buffer A. A linear gradient of NaCl (0–1.0 M) was used to elute the proteins. Fractions bearing xylanolytic activity were pooled, dialyzed in buffer A, and concentrated by ultrafiltration at 4,000 × g for 40 min at 4 °C using an Amicon Ultra Centrifugal Filter Device PL-10 . The fractions exhibiting enzyme activities were pooled and assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified recombinant TlXyn11B was deglycosylated by endo-β-N-acetylglucosaminidase H (Endo H)  at 37 °C overnight. The protein concentration was determined using the NanoVue Plus device  and Lowry protein assay37, with bovine serine albumin as the standard.Cells were removed by centrifugation, and the culture supernatants were subject to concentration through a Viva flow 200 ultrafiltration membrane . The crude enzyme was dialyzed in 20 mM citric acid-NaMS and MS/MS data for protein identification were obtained by using a MALDI-TOF-TOF instrument . The MS spectra were recorded in reflector mode in a mass range from 800 to 4,000 with a focus mass of 2,000. The TOF/TOF calibration mixtures (AB SCIEX) were used to calibrate the spectrum to a mass tolerance within 10 ppm. For MS calibration, autolysis peaks of trypsin  were used as internal calibrates, and the most intense ion signals (up to 10) were selected as precursors for MS/MS acquisition, excluding the trypsin autolysis peaks and the matrix ion signals.TlXyn11B.Peptide mass finger printing PMF and MS/MS queries were performed by using the MASCOT search engine 2.2  embedded into GPS-Explorer Software 3.6 (Applied Biosystems). A GPS Explorer protein confidence index ≥95% were used to align with the deduced protein sequence of 2HPO4  containing 1.0% (w/v) beechwood xylan. The amount of reducing sugar released was determined using the 3,5-dinitrosalicylic acid (DNS) method38. For assays of pH adaptability and stability, glycine-HCl , citric acid-Na2HPO4  and glycine-NaOH  were used. The optimal pH for TlXyn11B activity was determined at 65 °C and pH 1.5 to 6.0 for 10 min. The pH stability of TlXyn11B was determined by pre-incubating the enzyme  at 37 °C for 1 h without substrate and measuring the residual activities under standard conditions (pH 3.5 and 65 °C for 10 min). The optimal temperature for TlXyn11B activity was determined by performing the activity assay at 30 to 80 °C and at pH 3.5 for 10 min. Thermal stability was tested by pre-incubating the enzyme  at 50–70 °C for 30 min without substrate and monitoring the residual xylanase activities under standard conditions.The standard assay for xylanase activity was performed at 65 °C for 10 min in citric acid-NaTlXyn11B activity was determined in the presence of 1, 5 and 10 mM of NaCl, KCl, LiCl, MgSO4, CaCl2, HgSO4, AgNO3, ZnSO4, FeCl3, NiSO4, CuSO4, SDS and EDTA. The reaction systems without any chemical addition were treated as the control.The effect of different metal ions and chemical reagents on the Km and Vmax values of TlXyn11B were determined under standard conditions for 5 min with 1−10 mg/mL beechwood xylan as the substrate. The GraphPad Prism version 5.01  was used for data analysis with the Michaelis-Menten model. Each experiment was repeated three times.The 2HPO4 buffered beechwood xylan (pH 3.5) was treated by 2 U of TlXyn11B at 50 °C overnight. The enzyme was then removed from the reaction system through the Nanosep centrifugal 3 K device . Aliquots (100 μL) of 100-fold diluted hydrolysate samples were analyzed by high-performance anion-exchange chromatography (HPAEC-PAD)  equipped with a pulsed amperometric detector ICS-5000, a CarboPac PA100 guard column (4 × 50 mm) and an analytical column (4 × 250 mm). The xylo-oligosaccharides were eluted by 100 mM NaOH at the rate of 1 mL/min and at the temperature of 22 °C. Xylose, xylobiose, xylotriose, xylotetrose, xylopentose and xylohexose were used as the standards. Each experiment was repeated three times.One milliliter of citric-NaS3 was subject to saturated mutation. After thermostability screening in 96-well microplates, the mutant with greatest thermostability was selected for the second saturation mutation at D35. Mutants harboring different mutation sites were generated using overlap PCR with KOD neo Plus polymerase  and specific primers (Supplementary Table Tm) of TlXyn11B and its mutants were analyzed by using the DSC. The proteins were diluted in 10 mM citric acid-Na2HPO4 (pH 6.0) to approximately 0.25 mg/mL, and heated over the temperature range of 30 to 100 °C at a rate of 1 °C/min in Nano-DSC . The experiment was repeated twice.The melting temperatures  and conjugate gradient method  with CThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.Supplementary material


We observed that sufficiently high insulin (> 500 nM) with an optimal pulse period (~ 4 min) could make islets to produce synchronous Ca2+ oscillations. Glucose and insulin, which are key stimulatory factors of islets, modulate islet Ca2+ oscillations differently. Glucose increases the active-to-silent ratio of phases, whereas insulin increases the period of the oscillation. To examine the dual modulation, we adopted a phase oscillator model that incorporated the phase and frequency modulations. This mathematical model showed that out-of-phase oscillations of glucose and insulin were more effective at synchronizing islet Ca2+ oscillations than in-phase stimuli. This finding suggests that a phase shift in glucose and insulin oscillations can enhance inter-islet synchronization.Pancreatic islets can adapt to oscillatory glucose to produce synchronous insulin pulses. Can islets adapt to other oscillatory stimuli, specifically insulin? To answer this question, we stimulated islets with pulses of exogenous insulin and measured their Ca The initial glucose and insulin were set to G1 = 8.3 mM and I1 = 0 nM, and the rotators were equilibrated for 10 min. Afterward, we stimulated the rotators with a high glucose (G2 = 8.8 mM) and/or high insulin (I2 = 1000 nM) for 2 min with a 4-min period. We repeated the same simulation with a lower concentration (I2 = 100 nM) of insulin stimulus with a modified modulation factor ν = ω(I2)/ω(I1) = 4.6/5.3 ≈ 0.87 using the dominant periods, 2π/ω(I1) = 4.6 min and 2π/ω(I2) = 5.3 min for I1 = 0 and I2 = 100 nM insulin infusions. The differential equations were numerically integrated using the 2nd order Runge-Kutta method with a sufficiently small time step, Δt = 0.01 [We simulated the Ca rad/min . Then, et = 0.01 .t-test. The data in the text are presented as the means ± standard deviations. Findings were considered statistically significant at P < 0.01.Differences between unpaired observations were evaluated with Student’s S1 File2+ oscillations for constant insulin infusion  and alternating insulin infusion  were compressed as a single file, S1_File.zip, in which each imaging experiment was recorded as a text file where the first column represents time (sec) and the following columns represent detrended Ca2+ traces from multiple islets.All the time traces of detrended Ca(ZIP)Click here for additional data file.


All the compounds were characterized by 1H- and 13C-NMR spectroscopy and elemental analysis. New hybrid analogs were evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in order to know their potential for the prevention of Alzheimer's disease (AD). In coumarinyl thiazole series, compound 6b was found as the most active member against AChE having IC50 value of 0.87 ± 0.09 μM, while the compound 6j revealed the same efficacy against BuChE with an IC50 value of 11.01 ± 3.37 μM. In case of coumarinyl oxadiazole series, 11a was turned out to be the lead candidate against AChE with an IC50 value of 6.07 ± 0.23 μM, whereas compound 11e was found significantly active against BuChE with an IC50 value of 0.15 ± 0.09 μM. To realize the binding interaction of these compounds with AChE and BuChE, the molecular docking studies were performed. Compounds from coumarinyl thiazole series with potent AChE activity  were found to interact with AChE in the active site with MOE score of −10.19, −9.97, −9.68, and −11.03 Kcal.mol−1, respectively. The major interactions include hydrogen bonding, π-π stacking with aromatic residues, and interaction through water bridging. The docking studies of coumarinyl oxadiazole derivatives 11(a–h) suggested that the compounds with high anti-butyrylcholinesterase activity  provided MOE score of −9.9, −7.4, and −8.2 Kcal.mol−1, respectively, with the active site of BuChE building π-π stacking with Trp82 and water bridged interaction.In a continuation of our previous work for the exploration of novel enzyme inhibitors, two new coumarin-thiazole  Alzheimer's disease (AD), the most common cause of dementia, is a neurodegenerative disorder mainly characterized by progressive deterioration of memory and cognition  and coumarin-oxadiazole 11(a–h) hybrids were synthesized and evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity. Furthermore, the molecular docking studies on both series were also performed to explore their binding interactions.In the present study, two new coumarin-thiazole 6(a–o) and oxadiazoles 11(a–h) were prepared with the aim to identify new and potent inhibitors of acetylcholinesterase and butyrylcholinesterase. Coumarinyl thiazole derivatives 6(a–o) were accessed through a multi-component reaction approach which starts with the preparation of 3-(2-bromoacetyl)-2H-chromen-2-one (3)via base-catalyzed condensation of readily available starting materials  followed by bromination  and thiosemicarbazide (5) provided the title compounds 6(a–o) in good yields -thione conjugates 11(a–h), the central intermediate 3--2H-chromen-2-one (8) was prepared by the reaction of coumarinyl hydrazide (7) with carbon disulfide in ethanolic solution of KOH in good yield  and different  amines (10) gave the desired compounds 11(a–h) in good yields  and coumarinyl oxadiazoles 11(a–h), were screened for their inhibitory activity against AChE and BuChE by Ellman's method. All the assays were carried out at micromolar level using neostigmine and donepezil as standard inhibitors having IC50 values of 28.2 ± 2.01 and 7.23 ± 0.13 μM for AChE, whereas 16.1 ± 1.13 and 0.03 ± 0.003 μM for BuChE, respectively. The results obtained for both series 6(a–o) and 11(a–h) are summarized in Tables 50 values revealed that most of the synthesized compounds displayed potent and selective inhibition toward cholinesterases.The target compounds, coumarinyl thiazoles 6b of the coumarinyl thiazole series was found to be the strongest AChE inhibitor with an IC50 value of 0.87 ± 0.09 μM (Table 50 = 7.23 ± 0.12 μM). The strong inhibitory potential of 6b could be credited to the electron-donating amine group present at meta-position of the aryl ring. The introduction of a bromo group at the meta-position produced comparable results  to the neostigmine. A slight decrease in the inhibition (IC50 = 1.08 ± 0.84 μM) was observed in case of compound 6h having methoxy group at para-position but the inhibition was still 26-fold stronger than the standard neostigmine  to the donepezil , a slight decrease in the inhibition was observed in compounds 6j, 6n, 6o, and 6l as compared to the potent analogs, but the inhibition was still stronger compared to neostigmine.Among them, μM Table . This co(11a–h), compound 11a was found to be the most potent AChE inhibitor having IC50 value of 6.07 ± 0.23 μM (Table n-Bu) group as revealed by compound 11b (IC50 = 9.41 ± 0.55 μM). When these aliphatic groups were replaced by aromatic substitutions as in 11c–f, reduced inhibition was observed  and two phenyl groups (11h) were also found as moderate inhibitors of AChE with two- and three-fold higher inhibition as compared to neostigmine  directed a small decrease in the inhibition but the compounds were still several-folds more active than neostigmine  were excellent AChE inhibitors than the two reference drugs neostigmine and donepezil while coumarinyl oxadiazoles (11a–h) showed strong inhibition for BuChE than the standard neostigmine. All in all, the target analogs proved to be very potent inhibitors of cholinesterase and by considering their strong inhibitory potential, these heterocyclic hybrid compounds hold great potential for the development of new targets for AD therapy.In general, among the synthesized analogs, the compounds from coumarinyl thiazole series 6b, 6h, 6i, and 6k) represented better interaction with the active site of AChE (4EY7) with MOE score of −10.19, −9.97, −9.68, and −11.03 Kcal.mol−1, respectively, whereas oxadiazoles having high activity against butyrylcholinesterase  represented better interaction with BuChE (4BDS) with MOE score of −9.9, −7.4, and −8.2 Kcal.mol−1, respectively, as compared to reference ligands (neostigmine and donepezil) as shown in Table 6b (IC50 = 0.87 ± 0.09 μM) demonstrated conventional hydrogen bonding with Glu202 (1.48 Å) and Ser203 (1.69 Å) due to amino group of aryl ring and π-π stacking with Trp86, Trp286, and Tyr341 along with water bridging with Tyr337, Tyr124, and Ser125 as shown in Figure 6h (IC50 = 1.08 ± 0.84 μM) represented hydrogen bonding with Tyr72 (2.89 Å) and π-π stacking with Trp86, Trp286, and Tyr341 and Phe338 along with water bridging with Tyr72, Tyr124, and Thr83 as shown in Figure 6i (IC50 = 2.34 ± 1.43 μM) also demonstrated almost similar interactions (as with compound 6h) except the length of hydrogen bond with Tyr72 (2.93 Å). Compound 6k represented hydrogen bond with Tyr124 (2.21 Å), π-π stacking with Trp86 and Tyr341 (not with Trp286) and water bridging with Thr83, Ser125, and Asp74 as shown in Figure in-vitro results, molecular docking studies suggested that compounds 6b, 6h, 6i, and 6k represented better interaction than donepezil; (i) due to hydrogen bonding, (ii) due to extra π-π stacking with aromatic residues, and (iii) due to more interaction through water bridging as shown in Figure 6b (magenta) with reference ligand donepezil (yellow) was depicted in Figure 6b (magenta) was represented in Figure Possible binding modes of coumarinyl thiazole and oxadiazole derivatives were explored by MOE (Molecular Operating Environment) software. Molecular docking studies revealed that the thiazoles having high anti-acetylcholinesterase activity ( AChE 4EY with MOE11e (IC50 = 0.15 ± 0.09 μM) illustrated π-π stacking with Trp82 and interaction through water bridging with Asp70 and Ser79 as shown in Figure 11a (IC50 = 0.34 ± 0.06 μM) and 11b (IC50 = 0.77 ± 0.08 μM) represented conventional hydrogen bonding with Glu197 (1.5 Å) and Ser198 (3.0 Å), respectively as shown in Figures 11e (pink) with reference ligand donepezil (yellow) in the active site of BuChE (4BDS) ribbon model and surface model was depicted in Figures According to molecular docking results, the highest ranked anti-butyrylcholinesterase compound in-silico ADMET properties of lead molecules has now realized as an effective tool in the drug discovery and development process. Therefore, Lipinski's criteria and oral rat LD50 value were estimated for top ranked active compounds by using TEST (Toxicity Estimation Software Tool) and Molinspiration online software. Top five compounds selected from coumarinyl thiazoles and oxadiazole  for the analysis and results were summarized in Table 2 or less. Almost all the compounds fulfilled the criteria except compound 6b with cutoff value slightly higher i.e., 93. Number of rotatable bonds (nROT) is an additional property that measures the flexibility of the molecule. The drugs that are BBB +ve, usually reported to have fewer nROT bonds. Another extension in RO5 to improve the prediction of drug-likeness is molar refractivity (MR) which should be 40–130. Oral rat LD50 was also predicted for these compounds and the compounds were found slightly toxic according to Hodge and Sterner scale. All the criteria were fulfilled by the compounds and no Lipinski's violation was found as shown in Table Early prediction of 6b from coumarinyl thiazole series was emerged as the most potent inhibitor of AChE, whereas 11e from coumarinyl oxadiazole derivatives inhibited the BuChE with highest potency. Both the identified inhibitors follow Lipinski's RO5, slightly toxic and near the range of blood brain barrier crossing. In future, these compounds and their functionalized derivatives may be helpful in the development of potent drugs for Alzheimer's disease.In summary, the present report clearly revealed that the new hybrid molecules show remarkable inhibition of AChE and BuChE enzymes. Compound 6(a–o) and oxadiazole 11(a–h) analogs were prepared according to our recently published report  at pH 8, 10 μL of the test compound, 10 μL enzyme acetylcholine or butyrylcholinesterase of 0.03 U/mL (500 U of AChE and 700 U/mg of BuChE). The contents were incubated for 10 min at 25°C followed by the addition of 1 mM of 10 μL of substrate acetylcholine iodide for AChE and butyrylthiocholine iodide for BuChE and incubated again at 25°C for 15 min. A 50 μL of 3 mM DTNB as a coloring agent was added and incubated at 25°C for further 10 min. The amount of product formed was measured by using micro plate reader  at 405 nm. The enzyme dilutions were made by using buffer of pH 8 (tris base 50 mM and having 0.1% BSA). The compounds which depict inhibitory activity more than 50% were further tested by making 9–12 serial dilutions in assay buffer and IC50 values were calculated by graph pad prism.For the determination of cholinesterase inhibition, electric eel, and horse serum were used as source of AChE and BuChE, respectively. The Ellman's spectrophotometric method was used to determine the AChE and BuChE inhibitory activity with a slight modification  and butyrylcholinesterase (PDB: 4BDS) were retrieved from Protein Data Bank , and Tacrine (PDB: THA) were taken as possible binding site. Ligand neostigmine was taken from PubChem (CID:4456)  and converted into 3D conformations by ChemBio3D Mills, . MoleculAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Triptolide (TP), an active constituent of Tripterygium wilfordii, possesses numerous pharmacological activities. However, its effects on cytochrome P450 enzymes (CYP450s) in rats remain unexplored.In this study, the effects of triptolide on the six main CYP450 isoforms  were investigated both in vivo and in vitro. We monitored the body weight, survival proportions, liver index, changes in pathology, and biochemical index upon TP administration, in vivo. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we then carried out in vitro enzymatic studies in liver microsomal incubation systems via ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Finally, we verified our results at the messenger ribonucleic acid (mRNA) and protein level through quantitative real-time polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemical detection.The in vivo toxicity study confirmed that Sprague-Dawley (SD) rats exhibited dose-dependent hepatotoxicity after intragastric administration of TP  for 28 days. In case of the CYP450 isoforms 3A, 2C9, 2C19, and 2E1, the in vitro metabolic study demonstrated a decrease in the substrate metabolic rate, metabolite production rate, and Vmax, with an increase in the Km value, compared with that observed in the control group. Additionally, a TP dose-dependent decrease in the mRNA levels was observed in the four major isoforms of CYP3A subfamily  and CYP2C9. A similar effect was also observed with respect to the protein levels of CYP2C19 and CYP2E1.This study suggests that TP can cause hepatotoxicity by reducing the substrate affinity, activity, and expression at the transcriptional and protein levels of the CYP450 isoforms 3A, 2C9, 2C19, and 2E1. TP also has the potential to cause pharmacokinetic drug interactions when co-administered with drugs metabolized by these four isoforms. However, further clinical studies are needed to evaluate the significance of this interaction.The online version of this article (doi:10.1186/s12906-016-1504-3) contains supplementary material, which is available to authorized users. Tripterygium wilfordii Hook F (TWHF). It shows promising anti-inflammatory, immunomodulatory, anti-proliferative, proapoptotic, and neuroprotective activities  was used as the enzyme activity index.To estimate the kinetic parameters, the microsomes were incubated with different concentrations of the cocktail probe  using an Agilent ZORBAX RRHD Eclipse Plus C18 column  . The elution profile was composed of an initial isocratic step with water (0.1% formic acid): acetonitrile (ACN) (95:5) for 1.5 min and then increasing ACN to 30% over 4 min to separate the substrates and metabolites at 30 °C. The column was washed with 90% of ACN for 2.5 min followed by the equilibration of the column for 5 min with 5% ACN. The flow rate was 0.4 mL/min and the injection volume was 5 μL. An Agilent 6460 triple quadrupole-mass spectrometer with an electrospray ion source  was used for flow injection analysis with optimized fragmentor and source parameters. The optimized source parameters for MS analysis were drying gas temperature, 350 °C; gas flow, 10 L/min; nebulizer gas flow pressure, 35 psi; and capillary voltage, 4500 V.While implementing this experiment, we realized that human CYP3A subfamily isoforms, such as CYP3A4, CYP3A43, CYP3A7, etc., have several commercially available antibodies; however, there are no such antibodies for the main CYP3A subfamily isoforms  and CYP2C9 of rats. In order to validate the metabolic study and further probe the specific roles of the CYP3A subfamily and CYP2C9, we measured the mRNA expression of five major CYP3A subfamily isoforms  –28 and CThe total protein extract was prepared by diluting 50 μL liver microsome suspension with 1 volume of radioimmunoprecipitation assay (RIPA) lysis buffer , 0.5% sodium deoxycholic acid, 0.1% SDS, and 50 mM Tris-Cl, pH 7.5) for 15 min and then centrifuging at 13,000 rpm 4 °C for 10 min. Aliquots containing 20 μg of protein were separated by SDS-PAGE on a 10% gradient gel  and transferred to a PVDF membrane which was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST)  for 1 h. The membrane was then incubated with rabbit anti-rat CYP2C19 polyclonal antibody (1:2000 dilution), rabbit anti-rat CYP2E1 polyclonal antibody (1:1000 dilution) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:2000 dilution), overnight at 4 °C. After rinsing five times with TBST, for 5 min each time, at 20 °C, the membrane was incubated with horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin G (IgG) antibody . The immunoreactive bands were visualized using an enhanced chemiluminescence detection system  and quantified by densitometry using ImageJ 1.41  software.The tissue sections were incubated with rabbit anti-rat CYP2C19 polyclonal antibody (1:200 dilution) and rabbit anti-rat CYP2E1 polyclonal antibody (1:100 dilution) in blocking solution (0.4% (v/v) Triton X-100 and 3% (v/v) normal goat serum (NGS) in PBS) for 1 h. After incubation, the slices were washed four times, for 10 min each time, with 0.01 M PBS. Then, the sections were again incubated for 2 h with anti-rabbit antibody (1:500 dilution) at room temperature. After several PBS washes, the antibody complex was detected using a modification of the ABC system . The immunoreactive cells were stereologically quantified using Image-Pro Express 6  software. The selected areas were digitized with an Olympus DX45 microscope . The images were taken using an Olympus DP72 camera  at an original magnification of 400 × .t test and among multiple groups using one-way ANOVA followed by Tukey’s post hoc test. Data are expressed as Mean ± SD and P < 0.05 was considered statistically significant.SPSS version 19.0 for Windows  was used for all statistical analyses. All experiments were performed at least thrice. Comparisons were made between two groups using two-sided Student’s After one-week administration of TP, the rats began to exhibit restlessness by jumping up and down. Some rats showed reduced spontaneous activity and were less responsive to outside stimulations. During the second week, the groups which were administered 400 and 600 μg/(kg.day) of TP began showing symptoms of mortality. In the third and fourth week, the rats of the TP groups showed several symptoms of severe poisoning such as arched vertical hair, squinting or closed eyes, reduced activity, diarrhea, weight loss, and death. Of the rats that lived through the 28-day treatment period, 11 were from the 200 μg/(kg.day) group, 8 were from the 400 μg/(kg.day) group, and 6 were from the 600 μg/(kg.day) group. The number of deaths observed during the 28 days is presented in Fig. .Fig. 2GeP < 0.05 or P < 0.01), especially during the latest week of measurement, compared with the steadily increasing trend observed in the control group. Among the TP treated groups, the group which received with 600 μg/(kg.day) of TP demonstrated a dramatic weight loss compared with that observed in other groups (P < 0.01). These experimental results are shown in ) for 28 days, the rats showed significant histopathological changes, mainly including apparent liver cell degeneration of liver cell and inflammatory cell infiltration of inflammatory cells in the hepatic lobules and portal area, as shown in Fig. -g.P < 0.05 or P < 0.01) in the levels of ALT, AST, T-chol, Trig, and GLU and significant decrease (P < 0.05 or P < 0.01) in the levels of Tp and ALB were observed in the TP groups in comparison with that observed in the control group. Thus, most of the biochemical indicators changed in a TP dosage-dependent manner compared with the observed values in the control group. However, there was no significant difference in GLB levels  in the microsomal CYP450 content  transitions, optimized fragmentor voltage, collision energy, and selected reaction monitoring (SRM) pairs of each substrate and its metabolite are reported in Table Stability tests showed that samples remained stable even after being placed at room temperature for 24 h and being subjected to three repeated freeze-thaw cycles. In the precision test, the average standard deviation of three substances corresponding to the peak area was < 5%. The recovery rate test showed that both the absolute and method recovery rates were > 90%.P < 0.05 or P < 0.01) in TP-treated groups than in the control group  were also seen in the metabolite production rates in TP-treated groups in comparison with that observed in the control group ]. After oral administration of either the vehicle or TP ) for 28 days, the liver microsomes were incubated with the cocktail probe (CAF/D860/MT/DM/CLZ/MDZ: 15/20/10/10/5/2.5 μM) for 30 min. The final concentrations of the substrates and metabolites were detected by UHPLC-MS/MS. Although the metabolic rates of CYP1A2 and CYP2D6 remained the same, the rates of other CYP450s showed significant changes , in comparison with the control group values. The 200 μg/(kg.day) TP group exhibited an increase in the Km values of CYP2C19 (1.31 fold) and CYP3A (1.23 fold). Similarly, the 400 μg/(kg.day) TP group exhibited an increase in the Km values of CYP2C19 (1.30 fold), CYP3A (1.78 fold), CYP2E1 (1.45 fold), and CYP2C9 (1.59 fold). The 600 μg/(kg.day) TP group showed a more dramatic increase in the Km values of CYP2C9 (1.91 fold), CYP3A (2.08 fold), CYP2E1 (1.47 fold), and CYP2C19 (1.33 fold). The metabolite production rates followed a decreasing trend (P < 0.05 or P < 0.01) in the TP-treated groups in comparison to that observed in the control groups. While the metabolite production rates of CYP2C19 (87.8%) and CYP3A (58.8%) were decreased in the 200 μg/(kg.day) TP group, the rates of CYP2C19 (68.7%), CYP3A (77.5%), and CYP2C9 (75.4%) were decreased in the 400 μg/(kg.day) TP group. The 600 μg/(kg.day) TP group showed a more dramatic decrease in the metabolite production rates of CYP2C19 (60.9%), CYP3A (65.7%), CYP2C9 (60.0%), and CYP2E1 (85.9%) ] by incubating the microsomes with different concentrations of the substrates. The Km and Vmax values of each CYP450 isoform were calculated by non-linear regression analysis of experimental data according to the Michaelis–Menten model of enzyme kinetics, using the Prism 5.0 program. As shown in Fig. YP2C19 1.1 fold anThe Km value represents the affinity of a CYP450 enzyme for its specific substrate while the Vmax value represents the theoretical maximum metabolic rate of the substrate in the incubation system. These two parameters are very important indicators in the study of the kinetics of enzyme-catalyzed reactions. In comparison with the Km and Vmax value trends of CYP450s in the control groups, the trends observed in the TP-treated groups after the 28-day treatment period reflected the inhibitory effects of TP on CYP450 activity. Further, this TP-induced inhibition of CYP450 activity became more pronounced with the increase in TP dosage.P < 0.05 or P < 0.01) in comparison with that observed in the control group ) for 28 days, the mRNA levels of many CYP3A subfamily isoforms and CYP2C9 in TP-treated groups showed a significant decrease , in comparison to that observed in the control. The 600 μg/(kg.day) TP group exhibited a particularly strong decrease in CYP2C19 (41.9%) and CYP2E1 (51.4%) protein expression.When the effects of TP on CYP450 protein expression in rat liver microsomes were studied Fig. -c, TP waP < 0.05 or P < 0.01), in comparison to that observed in the control. The immunoreactive cells of the 600 μg/(kg.day) TP group exhibited a particularly strong decrease in CYP2C19 (27.5%) and CYP2E1 (38.2%) expression.Results of the immunohistochemical detection of CYP2C19 and CYP2E1 in rat liver sections were as shown in Fig. -e. TP waBased on pre-experimental observations, we found that TP-induced liver injury was more severe and stable in female rats than in male rats. Hence, female SD rats were specifically selected for this study. We found a certain correlation between the dosages of TP and hepatotoxicity in rats, which was consistent with previously published results , 21. TheAn intensive understanding of the CYP450 dysregulation phenomenon in TP-induced liver injury is necessary because of its significance in guiding the drug dosage in patients who require the co-administration of other drugs with TP. As shown in Fig. In our study, we observed a TP dose-dependent decrease in the mRNA expression of most CYP3A subfamily isoforms and CYP2C9, which was consistent with the results of our metabolic study. Among the five main CYP3A isoforms in rats, the expression of CYP3A2 and CYP3A62 mRNAs was most sensitive to TP-induced toxicity. The expression of CYP3A1/3A23 and CYP3A9 mRNAs was also significantly decreased at higher TP dosages, while there was no such inhibitory effect on CYP3A18. This phenomenon may have been due to the use of adult rats in this experiment, as CYP3A18 expression is strongly correlated with growth hormone production . CYP3A4 CYP2C9, an important component of the liver enzyme system, comprises about 10% of the total liver CYP450 enzyme content and is involved in the metabolism of 10% of the drugs in clinical trials . In thisAccording to our results, the protein expression of CYP2C19 and CYP2E1 decreased in a TP dose-dependent manner, which was consistent with the results of our metabolic study. Besides the CYP3A subfamily, CYP2C19 is also reported to be involved in TP metabolism and toxicity , 34, andThis experimental research demonstrated the abnormal metabolism of CYP450 enzyme system after TP administration. This results will hopefully serve as useful information for further understanding of the underlying mechanisms of TP-induced subacute toxicity. This results also suggests that when the patient needs to take TP for a certain time, doctors should pay attention to avoid the co-administration with drugs which metabolized by CYP3A, CYP2C9, CYP2C19, CYP2E1. If the co-administration was inevitable, doctors should closely observe the liver function.There are several limitations in our work. First, the relationship between CYP450 dysregulation and TP-induced hepatotoxicity remains undefined because we did not determine the exact mechanism through which TP affects CYP450 activity. Second, although we observed the effect of different concentrations of TP on CYP450 activity, the effect of different times of drug administration still needs to be investigated. Finally, all our experiments were conducted in rat liver microsomes or tissues; therefore, the effects of TP on real patients need to be investigated in the future.In summary, intragastric administration of TP ) for 28 consecutive days can cause hepatotoxicity by reducing the substrate affinity, activity, and expression at the transcriptional and protein level of CYP3A, CYP2C9, CYP2C19, and CYP2E1. TP also has the potential to cause pharmacokinetic drug interactions when co-administered with drugs metabolized by these four CYP450 isoforms. However, further clinical studies are needed to evaluate the significance of this interaction.


The effect of the bottom electrodes of Pt and TiN on the resistive switching properties of trilayer-structure units has been investigated. Both Pt/HfO2/TiO2/HfO2/Pt and Pt/HfO2/TiO2/HfO2/TiN exhibit typical bipolar resistive switching behavior. The dominant conduction mechanisms in low and high resistance states (LRS and HRS) of both memory cells are Ohmic behavior and space-charge-limited current, respectively. It is found that the bottom electrodes of Pt and TiN have great influence on the electroforming polarity preference, ratio of high and low resistance, and dispersion of the operating voltages of trilayer-structure memory cells. Compared to using symmetric Pt top/bottom electrodes, the RRAM cells using asymmetric Pt top/TiN bottom electrodes show smaller negative forming voltage of −3.7 V, relatively narrow distribution of the set/reset voltages and lower ratio of high and low resistances of 102. The electrode-dependent electroforming polarity can be interpreted by considering electrodes’ chemical activity with oxygen, the related reactions at anode, and the nonuniform distribution of oxygen vacancy concentration in trilayer-structure of HfO2/TiO2/HfO2 on Pt- and TiN-coated Si. Moreover, for Pt/HfO2/TiO2/HfO2/TiN devices, the TiN electrode as oxygen reservoir plays an important role in reducing forming voltage and improving uniformity of resistive switching parameters.The HfO Resistive random access memory (RRAM) has attracted great attention due to its potential for the replacement of flash memory in next-generation nonvolatile memories –3. Resis2O3/HfO2/Al2O3 exhibited fantastic uniformity of set and reset voltages, and excellent endurance of switching between the LRS and HRS 4 (TEMAH), TiCl4, and H2O as the Hf, Ti, and O precursors, respectively, where one oxide cycle consisted of 0.1s metal source injection, 4s N2 purging, 0.1s H2O injection, and 4s N2 purging. TEMAH was evaporated at 150 °C. Pure N2 (99.999%) was used as carrier gas and purging gas. Then, 100-nm-thick Pt top electrodes were DC sputtered through a shadow mask with a diameter of 150 μm using the Q150T system.Subsequently, 5 nm HfO2 or TiO2 on Si was determined by spectroscopic ellipsometer . The topography and surface roughness of the films and bottom electrodes were analyzed by atomic force microscopy . The root-mean-square (RMS) roughness values were recorded from 1 μm × 1 μm areas. The composition and chemical state of the stack structures were examined by X-ray photoelectron spectroscopy  with a monochromatic Al Kα source (hν = 1486.6 eV) for excitation of photoelectrons. The charge effect was calibrated by setting the C 1s photoemission at 284.6 eV. The XPS depth profile of HfO2/TiO2/HfO2 on Pt- and TiN-coated Si was obtained by Ar ion etching. The electrical properties of the HfO2/TiO2/HfO2 trilayer-structure RRAM devices were measured by Keithely 4200 semiconductor characterization system on probe station (CasCade Summit 12000 B-M). A current compliance of 10 mA was imposed to protect the fabricated device units from damages of high current during set processes. The bias voltage was applied to the Pt top electrode with the grounded bottom electrodes of Pt or TiN.The growth per cycle (GPC) of pure HfO2/TiO2/HfO2 trilayer-structure by ALD is illustrated in Fig. 2/TiO2/HfO2 on Pt- and TiN-coated Si have been examined. The Pt bottom electrode has smaller RMS value of 0.39 nm than PEALD-derived TiN of 0.87 nm. Hence, the sample of HfO2/TiO2/HfO2 on Pt-coated Si also exhibits relatively smoother surface with RMS of 0.68 nm than that on TiN-coated Si with 1.3 nm.The schematic of the RRAM device of HfO2/TiO2/HfO2/Pt and Pt/HfO2/TiO2/HfO2/TiN devices containing the initial electroforming process are plotted in Fig. 2/TiO2/HfO2/Pt device unit shows a forming voltage of +7 V in Fig. 5. The electroforming and rest process can also be completed by applying a negative voltage of −11 V and a positive one of +4 V, respectively, which are much larger than the positive forming and negative reset voltages. Moreover, the device cell only switches from LRS to HRS for several cycles after the negative forming process and then fails to reset to HRS due to the irreversible breakdown (not shown here). In Fig. 2. When imposing the positive electroforming voltage, the RS phenomenon cannot be observed in the Pt/HfO2/TiO2/HfO2/TiN cell and the device is permanently broken down at +14 V without the following efficient reset from LRS to HRS at negative voltage (not shown here).The DC I-V curves of Pt/HfO2/TiO2/HfO2 on Pt- and TiN-coated Si.The trilayer-structure RRAM devices with symmetric Pt top/bottom electrodes and asymmetric TiN bottom electrode/Pt top electrodes exhibit such different electroforming polarity preference. The bottom electrode of chemically inert Pt or relatively active TiN seems to play a key role. The related reason on electrode-dependent electroforming polarity will be discussed later after considering the XPS depth profiles of trilayer structures of HfO2/TiO2/HfO2/Pt and Pt/HfO2/TiO2/HfO2/TiN for 200 times tests, respectively. Figure 2/TiO2/HfO2/Pt and Pt/HfO2/TiO2/HfO2/TiN, respectively. The trilayer-structure RRAM device units with symmetric Pt top and bottom electrodes show a broad distribution for set voltage from 1.2 to 2.8 V and reset voltage from −0.5 to −1 V  of trilayer HfO2/TiO2/HfO2. Whereas, the metal ions in Al2O3 and HfO2 have different chemical valence of Al3+ and Hf4+, producing more interface charged defects of oxygen vacancies between two ILs of Al2O3/HfO2/Al2O3. It can be deduced that the interface O2− amount by adding two ILs between Al2O3/HfO2/Al2O3 should be higher than between HfO2/TiO2/HfO2. In addition, the formation energy of oxygen vacancy in storage layer of Al2O3/HfO2/Al2O3 is also higher than that of HfO2/TiO2/HfO2 (formation energy of oxygen vacancy 7.08 eV (Al2O3), 6.53 eV (HfO2), and 4.35 eV (TiO2) [2/TiO2/HfO2/TiN exhibits endurance degeneration, compared to Pt/Al2O3/HfO2/Al2O3/TiN.The endurance and retention properties of the device units of Pt/HfO105 Fig. a; howeve/Pt Fig. b. The ON in Ref. , the endous work , excelleV (TiO2) ). After 2/TiO2/HfO2/Pt and Pt/HfO2/TiO2/HfO2/TiN, the resistance level of the ON and OFF states has no evident changes after cumulative waiting time of 104 s at room temperature in Fig. For Pt/HfO2/TiO2/HfO2/TiN devices have much lower resistance ratio of OFF/ON states than Pt/HfO2/TiO2/HfO2/Pt, which can be attributed to the difference of the bottom electrodes in work function and conductivity. The work function of Pt and TiN bottom electrodes is 5.65 and 4.5 eV, respectively; hence, the interface barrier between TiN and underlying HfO2 is relatively lower in the Pt/HfO2/TiO2/HfO2/TiN device. During a reset process, the needed energy for electron to cross the barrier is also smaller. So Pt/HfO2/TiO2/HfO2/TiN manifests lower resistance value in OFF state than Pt/HfO2/TiO2/HfO2/Pt correspondingly. During a set process, RRAM device changes from HRS into LRS owing to the formation of the conducting channels. The Pt bottom electrode has higher conductivity than TiN bottom one; accordingly, the resistance value in ON state for Pt/HfO2/TiO2/HfO2/TiN is higher than that for Pt/HfO2/TiO2/HfO2/Pt. As a result, Pt/HfO2/TiO2/HfO2/TiN devices show a smaller resistance ratio of OFF/ON states of 102. However, the resistance ratio of 102 has already met the requirement of RRAM applications.Pt/HfO2/TiO2/HfO2/Pt and Pt/HfO2/TiO2/HfO2/TiN devices. Both memory cells behave similar conductive mechanism. When the device is switched to the LRS, the curves of log (I)-log (V) are linear with slope close to 1 . At critical voltage  functions after smart-type background subtraction.In order to further understand the underlying resistive switching mechanism of HfOf, Ti 2p, and O 1s peaks in HfO2 and TiO2 layers on TiN-coated Si. The Hf 4f and Ti 2p spectra from HfO2 and TiO2 layers can be deconvoluted into two group peaks. The stronger Hf 4f5/2 and Hf 4f7/2 peaks at 18.4 and 16.7 eV with a spin–orbit splitting of 1.7 eV are assigned to Hf–O bonding (Hf4+) from HfO2 , indicating the presence of oxygen vacancies in HfO2 layer. The calculated percentage concentration of Hfn+ (n < 4) is about 3.7% in Hf ions. In Fig. p3/2 and 2p1/2 features at 458.8 and 464.5 eV, belonging to the Ti–O bonding (Ti4+) from TiO2. A weaker doublet locates at 456.1 and 462.0 eV, assigning to the Ti3+–O bonding. The calculated percentage concentration of Ti3+ is about 21% in Ti ions. This implies the existence of oxygen vacancies in TiO2 layer.Figure s spectra from HfO2 and TiO2 layers can also be deconvoluted into two peaks, as seen in Fig. s peak at ~530.9 and 531.2 eV are assigned to Ti-O and Hf-O bonding in TiO2 and HfO2 layers, respectively, belonging to lattice oxygen without oxygen vacancies. Whereas the slightly higher energy of 532.1 eV in the O 1s spectra of Fig. 2 and TiO2 layers based on the literature reports [2 and TiO2 layers is about 2.3 and 19.4%, respectively, in accord with the results of Hfn+ and Ti3+.The O 1 reports –25. The  reports , 23. The2/TiO2/HfO2 samples on Pt- and TiN-coated Si by Ar ion etching, respectively. The trilayer-structure of HfO2/TiO2/HfO2 on Pt- and TiN-coated Si can be recognized easily, although the significant interfacial diffusion between HfO2/TiO2 and HfO2/TiN has been observed. Usually the filament model of oxygen vacancy migration dominates the RS behavior in RRAM devices based on transition metal oxides [Figure l oxides . Howeverl oxides . Lots ofl oxides , 26–29. l oxides , 17, 30.2/TiO2/HfO2 on Pt- and TiN-coated Si based on the above depth XPS profiles, respectively. The oxygen vacancy concentration was evaluated using the method mentioned above. It can be found that the highest oxygen vacancy concentration (~34%) appears in TiO2 intermediate layer. Another noteworthy feature is that the underlying HfO2 layer near Pt or TiN bottom electrodes has higher oxygen vacancy concentration than upper HfO2 layer (~2%). Besides, the oxygen vacancy concentration (~9%) of underlying HfO2 layer on TiN-coated Si is obviously higher than that (~6%) on Pt-coated Si.Figure 2/TiO2/HfO2/Pt at positive and negative bias voltages. For chemically inert Pt top and bottom electrodes, the bias polarity-dependent electroforming phenomena are related to the O2 gas released from the oxide layer at the anode as a product of electro-reduction. As known, during electroforming process, oxygen vacancies are created by high electric field and migrate to the cathode, forming localized conducting filaments in the oxide layer, i.e., OO→VO2+ + O2−, VO2+ + 2e−→VO [2− ions drift towards the anode, releasing their charge and evolving O2 gas, i.e., O2−→1/2O2 + 2e−  nearby Pt bottom electrode than the upper HfO2 layer (~2.3%)  Fig. c, the cob3% Fig. 2 layer and above Pt the bottom electrode  is lower than that of the underlying HfO2 (~6%)  Fig. c, the hiias Fig. g, the meunctions , 32.2/TiO2/HfO2/TiN devices exhibit quite different electroforming polarity from Pt/HfO2/TiO2/HfO2/Pt. The LRS caused by electroforming can be observed at both negative and positive bias voltage; however, the effective reset from LRS to HRS can be achieved only at positive bias voltage in Pt/HfO2/TiO2/HfO2/TiN device. Similar electroforming preference with asymmetric Pt and TiN electrodes has been observed in some literatures with various storage oxide layers such as HfO2 [2 [x/HfOy bilayer [2O3/HfO2/Al2O3 trilayer [Pt/HfO as HfO2 , 34, TiO HfO2 [2 , ZrOx/Hf bilayer , and Al2trilayer . However2/TiO2/HfO2 based on the XPS depth profiles  interfacial layer [2 layer into TiN bottom electrode has been confirmed in our sample by the XPS depth profile  and intermediate TiO2 layer (~10–34%) than the upper HfO2 layer (~1.9%), the migration of the rich oxygen vacancies from the underlying HfO2 layer through TiO2 layer towards the the upper HfO2 layer, directly producing conductive oxygen vacancy filaments with a smaller forming voltage of −3.7 V , which is beneficial to the reset operation from LRS to HRS with rupture of conductive filament  than the underlying HfO2 layer (~9%) and intermediate TiO2 layer (~10–34%), this leads to further increase of the oxygen vacancy concentration in underlying HfO2 and intermediate TiO2 layer. In the same time, due to slight oxygen existence in PEALD-derived TiN films (about 10%), some oxygen ions in TiN drift into underlying HfO2 layer to form insulate lattice oxygen with nearby oxygen vacancy, preventing from the growth of conductive filaments (Fig. 2/TiO2/HfO2/TiN device.On the other hand, when imposing the positive forming voltage on top electrode Pt, the oxygen vacancies migrate from the upper HfO HfO2 ~1.% than thnts Fig. . Therefo2 layer stores more oxygen vacancies than HfO2 layer, the distribution of oxygen vacancies in trilayer structure is not uniform, especially in the two interfacial layers between HfO2/IL/TiO2/IL/HfO2, which might affect the growth position, direction, and overlapping of conductive filaments. The linkage or rupture of the conductive filaments corresponds to the set process from HRS to LRS or the reset process from LRS to HRS, respectively, which more easily happens in two interfacial layers. Further, the shape and position of the conductive filaments in HfO2 and TiO2 layers change less in the set and reset processes.Finally, on account of the fact that the inserted TiO2/TiO2/HfO2/Pt and Pt/HfO2/TiO2/HfO2/TiN have been prepared by ALD. Both memory cells show typical bipolar resistive switching characteristics, and Ohmic and SCLC dominant conduction mechanisms in LRS and HRS, respectively. It is found that the bottom electrodes of Pt and TiN have great influence on the electroforming polarity preference, the ratio of high and low resistances and dispersion of the operating voltage of trilayer-structure memory cells. Compared to with symmetric Pt top/bottom electrodes, the RRAM cells with asymmetric Pt top/TiN bottom electrodes show smaller negative forming voltage of −3.7 V, relatively narrow distribution of the set/reset operation voltages and lower ratio of high and low resistances of 102. The electrode-dependent electroforming polarity can be explained by considering electrodes’ chemical activity with oxygen, the related reactions at anode, and the nonuniform distribution of oxygen vacancy concentration in trilayer-structure of HfO2/TiO2/HfO2 on Pt- and TiN-coated Si. Furthermore, the TiN electrode as oxygen reservoir plays an important role in forming voltage reduction and better dispersion of RS parameters for Pt/HfO2/TiO2/HfO2/TiN devices. Considering the modulation effect of electrode and trilayer-structure on resistive switching performance, this work provides a new device design route for future RRAM applications.In summary, RRAM devices based on trilayer-structure of Pt/HfO


Mycobacterium tuberculosis, is the biggest infectious disease killer globally. New drugs for the treatment of the drug-resistant forms of the disease are needed. Recently, a new target-lead couple, the mycobacterial protease ClpP1P2 and the human anticancer drug bortezomib, was identified. However, we know little about how expression of this protease is regulated, which proteins in the bacterium it degrades, how the protease recognizes its target proteins, and how the inhibition of ClpP1P2 exerts whole-cell antimicrobial activity. Here, we show that the ClpP1P2 protease regulates its own expression, and we identified a new substrate and a new substrate recognition sequence and a mechanism for how ClpP1P2 inhibition causes bacterial growth inhibition.With 9 million new cases and more than 1 million deaths per year, tuberculosis, caused by  clpP1P2 genes and also suggested that this transcription factor may be a substrate of the protease. Here, we employ promoter activity reporters and direct mRNA level measurements showing that bortezomib treatment of Mycobacterium bovis BCG increased transcription of clpP1P2 and other ClgR-dependent promoters, suggesting that inhibition of ClpP1P2 increases cellular ClgR levels. Then, we carried out red fluorescent protein-ClgR fusion analyses to show that ClgR is indeed a substrate of ClpP1P2 and to identify ClgR’s C-terminal nonapeptide APVVSLAVA as the signal sufficient for recognition and efficient protein degradation by ClpP1P2. Interestingly, accumulation of ClgR appears to be toxic for bacilli, suggesting a mechanism for how pharmacological inhibition of ClpP1P2 protease activity by bortezomib translates into whole-cell antibacterial activity.The mycobacterial caseinolytic protease ClpP1P2 is a degradative protease that recently gained interest as a genetically and pharmacologically validated drug target for tuberculosis. The first whole-cell active ClpP1P2 inhibitor, the human proteasome inhibitor bortezomib, is currently undergoing lead optimization to introduce selectivity for the bacterial target. How inhibition of ClpP1P2 translates into whole-cell antimicrobial activity is little understood. Previous work has shown that the caseinolytic protease gene regulator ClgR is an activator of the IMPORTANCE With 9 million new cases and more than 1 million deaths per year, tuberculosis, caused by Mycobacterium tuberculosis, is the biggest infectious disease killer globally. New drugs for the treatment of the drug-resistant forms of the disease are needed. Recently, a new target-lead couple, the mycobacterial protease ClpP1P2 and the human anticancer drug bortezomib, was identified. However, we know little about how expression of this protease is regulated, which proteins in the bacterium it degrades, how the protease recognizes its target proteins, and how the inhibition of ClpP1P2 exerts whole-cell antimicrobial activity. Here, we show that the ClpP1P2 protease regulates its own expression, and we identified a new substrate and a new substrate recognition sequence and a mechanism for how ClpP1P2 inhibition causes bacterial growth inhibition. One  chamber .in vitro- and in vivo-validated targets in Mycobacterium tuberculosis (–50)-MIC50 structure-activity relationship correlation studies, and structural analyses that it is indeed on the target, i.e., via inhibition of the ClpP1P2 protease (and not other cellular targets), that bortezomib exerts its antibacterial whole-cell activity  expression in the respective Mycobacterium bovis BCG reporter strains  and showed white colonies with minimal signals of RFP fluorescence  colonies were slightly pink, whereas M. bovis BCG RFP-ClgR(C9) colonies were white. Correspondingly, M. bovis RFP-ClgR(C5) broth cultures showed high levels of fluorescence, an indication of poor degradation of the RFP carrying only the last 5 amino acids of ClgR. In contrast, cultures of M. bovis RFP-ClgR(C9) showed a background level of fluorescence, similarly to cultures of M. bovis BCG expressing the RFP–full-length ClgR fusion, indicating efficient degradation of RFP-ClgR(C9) fusion protein  were not successful, i.e., nstructs . SimilarM. bovis BCG strain overexpressing the full-length ClgR protein (containing the Clp recognition sequence) could be constructed. In contrast, when we transformed plasmids overexpressing C-terminal truncations of ClgR (lacking the Clp recognition sequence), we were unable to obtain transformants  to incrhttp://www.newtbdrugs.org/pipeline/discovery) may cause inhibition of growth of the organism.In conclusion, we first provide evidence that the mycobacterial ClgR is a substrate of ClpP1P2. Thus, we identified a novel regulatory feedback loop in mycobacteria: ClpP1P2 controls its own expression by regulating the level of the transcriptional activator of its encoding genes. Second, we identified the C-terminal nonapeptide APVVSLAVA of ClgR as a novel degradation tag recognized by Clp. Finally, we show that accumulation of ClgR appears to be toxic for the bacteria, and thus we provide a mechanism for how inhibition of ClpP1P2 by the new lead compound bortezomib (M. bovis BCG Pasteur ATCC 35734 (BCG) was purchased from the American Type Culture Collection and was grown at 37°C in Middlebrook 7H9 broth (BD Difco) supplemented with 0.5% albumin, 0.2% glucose, 0.085% sodium chloride, 0.0003% catalase, 0.2% glycerol, and 0.05% Tween 80. Genomic DNA was isolated from M. bovis BCG as described previously (Tables S1 and 600) reached 0.2, 0.1 volume of 2 M glycine was added, and cells were further incubated for 16 h. Cells were washed 3 times with wash buffer (10% [vol/vol] glycerol and 0.05% Tween 80 in Milli-Q H2O) and resuspended in 0.02 volume of the initial culture. Electrocompetent BCG cells were mixed with 100 ng of plasmid, and electroporation was performed with a Gene Pulser apparatus (Bio-Rad) at 2,500 V, a capacity of 25 µF, and a resistance of 1,000 Ω. Bacteria were cultured overnight in fresh 7H9 and plated on 7H11 agar (BD Difco) with 25 µg/ml of kanamycin.eviously . Primers10.1128/mSphere.00338-16.1TABLE S1 TABLE S1, PDF file, 0.1 MB.Reporter plasmids for measuring promoter activities and primers used in this study. Download Copyright © 2017 Yamada and Dick.2017Yamada and DickCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00338-16.2TABLE S2 TABLE S2, PDF file, 0.1 MB.Primers used for qRT-PCR. Download Copyright © 2017 Yamada and Dick.2017Yamada and DickCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00338-16.3TABLE S3 TABLE S3, PDF file, 0.1 MB.Plasmids to overexpress recombinant proteins and primers used in this study. Download Copyright © 2017 Yamada and Dick.2017Yamada and DickCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 600), and relative fluorescence units  by using an Infinite M200 Pro plate reader (Tecan). Briefly, log-phase (OD60 of 0.4 to 0.6) M. bovis BCG cultures were adjusted to an OD600 of 0.4 in fresh 7H9, and 100 µl of cell suspension was inoculated into 96-well microplates which contained an equal volume (100 µl) of fresh 7H9 with or without drugs. After the measurement at day 0, microplates were sealed with Breathe-Easy membrane (Sigma-Aldrich) and incubated at 37°C with shaking at 80 rpm for 24 h.Reporter assays were carried out with dual readout, absorbance , and subjected to bead beating by using a FastPrep-24 5G instrument . RNA was purified using the PureLink RNA minikit with the Turbo DNA-free kit (Invitrogen). cDNA was created from 4 µg of total RNA with the SuperScript III first-strand synthesis system (Invitrogen) by using random primers. Quantitative PCR was performed with the FastStart Essential DNA Green Master (Roche) using the LightCycler 96 real-time PCR system (Roche).RNA from


Background: Model-based compartmental analysis of data on plasma retinol kinetics after administration of labeled retinol provides unique information about whole-body vitamin A metabolism. If labeled β-carotene is coadministered, its bioefficacy relative to the retinol reference dose can also be estimated.Objectives: The objectives were to model plasma retinol kinetics after administration of labeled preformed vitamin A and provitamin A β-carotene and to determine relative β-carotene bioefficacy.Methods: We used the Simulation, Analysis and Modeling software  to analyze previously collected data on plasma [13C10]- and [13C5]retinol kinetics for 14 d after oral administration of 1 mg [13C10]retinyl acetate and 2 mg [13C10]β-carotene in oil to 30 healthy young adults of European ancestry  with moderate vitamin A intakes.Results: A 6-component model provided the best fit to the data, including compartments for initial metabolism of vitamin A, plasma retinol, and extravascular vitamin A storage. The disposal rate was 6.7 ± 3.1 μmol/d, fractional catabolic rate was 6.0% ± 2.3%/d, and vitamin A stores were 123 ± 71 μmol. Relative β-carotene bioefficacy, based on the ratio of the areas under the fraction of dose curves calculated by WinSAAM, averaged 13.5% ± 6.02% . Interindividual variation in relative β-carotene bioefficacy was high (CV: 44%).Conclusions: Vitamin A kinetics in these young adults were best described by essentially the same model that had been previously developed by using data for older adults with higher vitamin A stores; differences in parameter values reflected differences in vitamin A status. Estimated β-carotene bioefficacy was relatively low but similar to previously reported estimates obtained by graphical methods. This trial was registered at the UK Clinical Research Network as UKCRN 7413. Results were based on plasma [2H8]retinol data collected for 56 d after the oral administration of deuterated retinyl acetate. Both groups in that study had moderate vitamin A stores, unlike the case in many lower-income populations worldwide for whom vitamin A deficiency and/or low vitamin A status are important public health problems.Model-based compartmental analysis  has beeAAM.org) ]. This aAAM.org) . In a puAAM.org)  presenteIn addition to information on vitamin A kinetics, WinSAAM can also be used to estimate relative β-carotene bioefficacy if a stable isotope of β-carotene is coadministered with labeled retinol. β-Carotene bioefficacy is defined as the proportion of ingested β-carotene that is absorbed and converted to retinol . The bio13C10]β-carotene and [13C10]retinyl acetate in oil. Our goals were to extend the knowledge on whole-body vitamin A metabolism in a population not previously studied and to compare model-generated estimates of β-carotene bioefficacy with graphical estimates. Because participants in the current study were selected to have moderate vitamin A intakes and were found to have relatively low vitamin A stores, our results may be generalizable to populations in lower-income countries who may be at risk of vitamin A deficiency.In the current study, we applied model-based compartmental analysis to the data of Oxley et al.  on plasm13C10]retinyl acetate  and [13C10]β-carotene  were purchased from Buchem BV. The isomeric composition of purchased [13C10]β-carotene was retrospectively determined to be 88.5% all-trans-β-carotene, 10.0% cis-β-carotene, and 1.5% α-carotene.As reported by Oxley et al. , [13C10]2) >30, high blood pressure, diabetes, lipid metabolic disorders, or liver, kidney, or gastrointestinal disease ; or consuming a diet high in preformed vitamin A (as determined by an FFQ). For the current modeling analysis, 15 of the 45 participants from the Oxley et al. study ; thus, subjects received 2.954 μmol [13C10]retinyl acetate (“[13C10]retinol dose”) and 3.237 μmol [13C10]all-trans-β-carotene, 0.366 μmol [13C10]cis-β-carotene, and 0.055 μmol [13C10]α-carotene (“β-carotene dose”). Knowing that 1 molecule of all-trans-β-carotene could maximally be absorbed and converted into 2 molecules of retinol, whereas cis-β-carotene or α-carotene could yield only 1 retinol molecule, the value for the [13C10]β-carotene dose was calculated to be 6.895 μmol retinol activity equivalents (RAEs) . Blood was collected into 10-mL EDTA-coated evacuated tubes before administration of the dose (0 h) and at 2, 4, 6, 8, 10, and 12 h after dosing; cannulas were then removed. Subsequent fasting blood samples were obtained by venipuncture at 1, 2, 7, and 14 d. Blood samples were centrifuged at 1300 × g for 10 min at 4°C immediately after collection; plasma was stored at –80°C until analysis.For the kinetic study, volunteers consumed a standardized pasta meal, along with water, the evening before the experiment started and then fasted overnight. In the morning, a cannula was inserted in an antecubital vein in each subject. Subjects were sequentially given the doses of [13C10]retinyl acetate and [13C20]β-carotene as internal standards. Solvent extracts were dried under a gentle stream of nitrogen, and the residues were reconstituted in 100 μL ethyl acetate. Analytes were separated by reverse-phase HPLC before MS/MS quantitation under atmospheric pressure chemical ionization in positive ion mode. Selected reaction monitoring of analytes was performed at m/z 547→330 and m/z 274→98 for [13C10]β-carotene and [13C5] cleavage products and at m/z 279→100 for metabolites of [13C10]retinyl acetate. [12C]Retinol in plasma was quantified by HPLC  according to the method of Oxley et al. . Briefly by HPLC .13C5]retinol (derived from [13C10]β-carotene) and [13C10]retinol (derived from [13C10]retinyl acetate) at each time for each subject was calculated as follows:The fraction of the oral dose (FD) in plasma for both [13C10]retinol and [13C5]retinol FD, background MS/MS analytical noise was subtracted for each sample.where estimated plasma volume (L) = body weight (kg) × 0.0435 (L/kg) . To accu13C10]retinol FD vs. time were fit to a model modified from the one developed by Cifelli et al.  and the fractional transfer of retinol back to plasma from stores is L (d−1). Parameters were systematically adjusted in WinSAAM to obtain a satisfactory fit of the data to the model for each subject. Final estimates for the Ls and their statistical uncertainties were generated by using weighted nonlinear regression analysis in WinSAAM. A fractional standard deviation (FSD) of 0.05 was used as the weighting factor.By using WinSAAM , the dati et al.  to descrnt model . Compart12 C]retinol concentration during the study (determined by HPLC) and their estimated plasma volume. Then, plasma retinol pool sizes were used in a steady state solution in WinSAAM to estimate vitamin A pool sizes or traced mass ; disposal rate (μmol/d), or the rate of loss of vitamin A from compartment 6; and system fractional catabolic rate , or the fraction of retinol in compartment 6 that leaves irreversibly each day. Other parameters that were calculated include the half-life [t1/2(J)] for retinol in compartments 5 and 6, where t1/2(J) = ln 2/∑Ls exiting compartment J. The mean residence time [T] is the mean time the tracer spends in compartment I after entering the system via compartment J; thus, residence time in plasma for absorbed retinol is T and residence time in stores is T. T(SYS) [T + T] is the total time the tracer spends in plasma and stores from the time it enters plasma until it irreversibly leaves the system. The recycling number [v(5)] is the average number of times a retinol molecule recycles to plasma before irreversible loss; v(5) = {[T/t1/2(5)] – 1}. Finally, the recycling time to plasma [tt(5)] was calculated as [T/v(5)].The following kinetic parameters were then calculated by using the Ls for each individual [see Cifelli et al.  and Gree13C10]retinol, a parallel model was formulated (see Results) for plasma [13C5]retinol  and WinSAAM was used as above to calculate additional model parameters as well as relative β-carotene bioefficacy.Once the data had been modeled for . We also calculated relative bioefficacy by using the graphical method described by Tang et al. , in whicP < 0.05 was considered significant. Student’s paired t test and linear least-squares regression analysis were used to compare β-carotene bioefficacy calculated by using WinSAAM compared with KaleidaGraph. Pearson’s correlation coefficient (r) and Spearman’s rank correlation coefficient (Rs) were determined.Data are presented as means ± SDs. Graphs were made by using GraphPad Prism , and statistical analyses were performed by using JMP Pro . n = 30) were healthy young adults of European ancestry (13 men and 17 women) with a mean ± SD age of 24.5 ± 4.2 y, mean ± SD body weight of 65.2 ± 10 kg, and mean ± SD BMI of 22.5 ± 1.9. The recruitment strategy selected volunteers with moderate intakes of preformed vitamin A, as determined by using the EPIC (European Prospective Investigation into Cancer and Nutrition) FFQ (Subjects (ion) FFQ . As show1) data were missing at critical time points (n = 1) or the FD was negative after background subtraction (n = 3) or 2) plasma isotope response curves did not follow the expected pattern of steady state tracer kinetics as evidenced by a positive terminal slope for [13C5]- and [13C10]retinol curves (n = 3), the 2 curves converged (n = 3) or diverged (n = 1) over time, or there was an abnormally high peak value for [13C10]retinol (n = 4).Data for 15 other subjects studied by Oxley et al.  were exc13C10]retinol vs. time for 1 representative subject are plotted in 13C10]retinol peaked at ∼12 h and then fell as tracer was distributed to extravascular tissues. The bend in the curve at ∼2 d indicates tracer recycling to plasma; the curve then enters a terminal slope, reflecting the system FCR for vitamin A. When fitting the data to the model shown in Data for FD in plasma retinol (derived from the [13C10]β-carotene dose) vs. time in the same subject as plotted in the upper curve. Note that the 2 curves appear parallel from ∼12 h after dosing; this was true for all subjects in the current analysis.The lower curve in 13C10]retinol data, we developed a parallel model for plasma [13C5]retinol  = L, L = L, and L = L. In the parallel model, compartment 11 represents β-carotene transit and absorption into enterocytes, and compartment 12 comprises the portion of absorbed β-carotene converted to retinol. Because a wide range of absorption and bioconversion efficiencies have been reported for β-carotene  = 3.6 h . As in tP = 0.23). Least-squares linear regression analysis .Mean β-carotene bioefficacy calculated by using WinSAAM was 13.5% ± 6.02% (range: 4–32%) or 7.7:1.0 μg RAEs. There was extensive interindividual variation in β-carotene bioefficacy (CV: 44%). The subject whose data are plotted in n = 30) of healthy young adults (mean age: 24 ± 4 y) of European ancestry. We found that the structure of the 6-component model developed by Cifelli et al. (n = 14) and 59 ± 9 y (n = 12), respectively] provided a good fit to the current data, with a minor modification needed in the site of loss of the unabsorbed oral doses and with adjustment of the parameter values. Modeling revealed that total body stores of vitamin A were lower (123 ± 71 μmol) in the current Newcastle volunteers than in the Chinese (233 ± 109 μmol) or American (892 ± 637 μmol) subjects. Although the Newcastle subjects were selected for their low to moderate vitamin A intakes, our analysis further underscores their relatively low vitamin A status, in that they had only 18 d of vitamin A stores (compared with 42 d in the Chinese and 61 d in the Americans), where “days of stores” was calculated as model-predicted micromoles of total body stores/disposal rate (μmol/d). Note that, as was the case in the Chinese and American subjects, plasma retinol concentrations were normal in the Newcastle volunteers (1.54 ± 0.28 μmol/L). That is, there was a high prevalence of low total body stores of vitamin A in the Newcastle cohort despite normal plasma retinol concentrations. Similar findings of low vitamin A stores and normal plasma retinol concentrations have been reported in both adult Bangladeshi men [60 μmol  = 4.8 d] than that determined for individuals studied by Cifelli et al. , 19. The13C10]retinol (from the [13C10]retinyl acetate dose) and [13C5]retinol (from the [13C10]β-carotene dose) were the same once retinol entered plasma bound to RBP. That is, based on visual inspection, the plasma response curves were parallel from ∼12 h after dosing (13C] atoms (10 compared with 5) in retinol from the 2 stable isotope doses.It is worth mentioning that, in the current subjects, the kinetics of β-carotene in oil  and those determined for the American and Chinese subjects studied by Tang and colleagues , we found that estimates generated by graphical analysis as described by Tang et al.  were conlleagues , 7 (11% lleagues , 23. Beclleagues , as welllleagues , 24, 25,In conclusion, the application of model-based compartmental analysis has the potential to provide unique information and insights into vitamin A and β-carotene kinetics and metabolism as well as practical results with the potential for application in the field. For example, in the accompanying article in this issue , we used