Glycyrrhizae radix et rhizoma has been used as a traditional Chinese medicine for the treatment of various diseases. Triterpenoids and flavonoids from the plant have many beneficial effects and their chemical structures are modified in the gastrointestinal tract after oral administration. However, absorption of these triterpenoids and flavonoids still needs to be defined. Here, the uptake and transepithelial transport of the selected major triterpenoids, glycyrrhizin (1), glycyrrhetic acid-3-O-mono-β-d-glucuronide (2), and glycyrrhetinic acid (3); and the selected major flavonoids, licochalcone A (4), licochalcone B (5), licochalcone C (6), echinatin (7), isoliquiritin apioside (8), liquiritigenin (9), liquiritin apioside (10) isolated from Glycyrrhizae radix et rhizoma, were investigated in the human intestinal epithelium-like Caco-2 cell monolayer model. Compounds 3, 5–7, and 9 were designated as well-absorbed compounds, 2 and 4 were designated as moderately absorbed ones, and 1, 8, and 10 were assigned for the poorly absorbed ones. The absorption mechanism of well and moderately absorbed compound was mainly passive diffusion to pass through the human intestinal Caco-2 cell monolayer. These findings provided useful information for predicting their oral bioavailability and the clinical application. Glycyrrhiza consists of about 30 species with a nearly global distribution, of which about 18 species are found in China. Among them, only three species—named Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., and Glycyrrhiza glabra L.—have been used as traditional Chinese medicines for the treatment of hepatitis, spasmodic cough, gastric ulcer, etc. [Glycyrrhiza [1), glycyrrhetic acid-3-O-mono-β-d-glucuronide (2) and glycyrrhetinic acid (3), and seven flavonoids; licochalcone A (4), licochalcone B (5), licochalcone C (6), echinatin (7), isoliquiritin apioside (8), liquiritigenin (9), and liquiritin apioside (10), whose chemical structures are shown in Glycyrrhizae radix et rhizoma, in Caco-2 cell monolayer model [The genus cyrrhiza ,3,4,5. Acyrrhiza ,7,8, antcyrrhiza ,10,11,12cyrrhiza ,14, hepacyrrhiza ,16, anticyrrhiza ,20,21,22cyrrhiza ,24,25, acyrrhiza ,27,28, acyrrhiza  propertier model ,32, and 2 were used for transport assays [Papp) values of propranolol and atenolol, the well- and poor-transported markers by passive diffusion mechanism across Caco-2 cell monolayer, were assessed as (2.51 ± 0.21) × 10−5 cm/s and (2.35 ± 0.11) × 10−7 cm/s, respectively. The results were in a good agreement with the reported values [H-tetrazolium bromide (MTT) assays [The integrity of differentiated Caco-2 cell monolayer was examined by measuring the transepithelial electrical resistance (TEER) with an epithelial voltohmmeter . Only cell monolayers with a TEER value above 500 Ω·cmt assays ,32. The y) versus concentration . Regression equations, coefficient correlations (r2), and concentration ranges of the calibration curve for the test compounds were listed in 1, 8, and 10 were not performed, because they were extremely difficult to transport through Caco-2 cell monolayer.The high performance liquid chromatography (HPLC) analysis methods for the all test compounds had been validated. The standard calibration curves were constructed by plotting peak area  × 10−5 cm/s), a well-transported marker of the transcellular pathway [3 was designated as a well-absorbed compound. The Papp values of compound 2 in the bidirectional transportation were of 1 × 10−6 cm/s magnitude, which lay between that of propranolol and atenolol, so compound 2 was designated as a moderately absorbed compound.The bilateral →basolateral side (BL) and BL→AP)  pathway , so comp3 is a triterpenoid aglycone, while compounds 1 and 2 are C3-O-β-d-bisglucopyranuronoside and C3-O-β-d-monoglucopyranuronoside of 3  of compounds 2 and 3 suggested passive diffusion as the main transport mechanism. It has been demonstrated in many reports that compound 1 shows substantial pharmacological effects [1 can be transformed to compounds 2 and 3 by intestinal bacteria following the oral intake of licorice in humans [2 and 3 were absorbed in blood as the active metabolite of licorice, especially compound 3.Compound ide of 3 , respectn humans . Therefo2 and 3 increased approximately linearly with time (0–180 min) at 50 μM (2 and 2.5–75 μM for compound 3). From kinetic curves presented in 2 and 3 decreased approximately linearly in AP side and increased approximately linearly in BL side within the incubation time.The bilateral permeation of compounds at 50 μM , and theat 50 μM  within tPapp values of chalcones 4–8 are summarized in 5–7 were designated as well-absorbed compounds, while chalcones 4 and 8 were designated as a moderately and poorly absorbed compounds, respectively. The Papp values of chalcone 11 (isoliquiritigenin) were reported to be (1.46 ± 0.01) × 10−5 cm/s from AP to BL direction and (9.35 ± 0.43) × 10−6 cm/s from BL to AP direction [12 (isoliquiritin) were reported to be (8.69 ± 0.15) × 10−7 cm/s from AP to BL direction and (7.68 ± 0.66) × 10−7 cm/s from BL to AP direction in our previous papers [Papp values of chalcone glycosides  were 1 × 10−7 cm/s or below, they were designated as the poorly absorbed compounds.The bidirectional on and 9. ± 0.43 ×s papers . The res4–7 increased approximately linearly with time (0–180 min) at 50 μM . From kinetic curves presented in 4–7 decreased in AP side and increased in BL side approximately linearly within the incubation time.The bilateral permeation of the chalcones at 50 μM , while tat 50 μM  within tPapp values of flavonones 9 and 10 are summarized in 9 was assigned as a well-absorbed compound with Papp value of around 1 × 10−5 cm/s. Flavonone 10 was found to hardly permeate Caco-2 cell monolayer with Papp value < 1 × 10−7 cm/s. The Papp values of flavonone 13 (liquiritin) were reported to be (5.40 ± 0.16) × 10−7 cm/s from AP to BL direction and (5.32 ± 0.31) × 10−7 cm/s from BL to AP direction in our previous study [9 > 13 > 10, i.e., disaccharide glycoside (10) showed less permeability than monosaccharide glycoside (13), and both of them had far less permeability than the corresponding aglycone (9). This was in accordance with the finding in the case of triterpenoids 1–3 that the permeability of compounds in the Caco-2 cell monolayer can be reduced with the increase in the number of glycosyl groups on the core molecular nuclei.The bidirectional us study , and it 9 increased approximately linearly with time (0–180 min) at 50 μM , as well as their MW values were shown in Papp AP→BL × MW1/2) versus logD was plotted  and MW (molecular weight) were generally utilized for the prediction of the permeability of compounds . An anal plotted  with Ori1), glycyrrhetic acid-3-O-mono-β-d-glucuronide (2), and glycyrrhetinic acid (3) were supplied by Natural Product Sample Library in State Key Laboratory of Natural and Biomimetic Drugs of Peking University . Chalcones licochalcone A (4), licochalcone B (5), licochalcone C (6), and echinatin (7) were obtained from Tianjin Chroma-standard Medical Science & Technology Development Co. Ltd. , and isoliquiritin apioside (8) was obtained from Shanghai Tauto Biotech Co. Ltd. . Flavonones liquiritigenin (9) and liquiritin apioside (10) were obtained from Shanghai Tauto Biotech Co. Ltd. .Triterpenoids glycyrrhizin .HPLC grade methanol (MeOH) was purchased from Fisher Scientific . Analytical grade acetic acid and Hank’s Balanced Salts Solution (HBSS) were purchased from Beijing Chemical Works . Water was purified by a Mili-system . Propranolol, atenolol and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co. . Penicillin-streptomycin solution was obtained from Suolaibao Technology Ltd. . Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), nonessential amino acids (NEAA), and trypsin were supplied by Gibco2–7 and 9 was performed on a Dionex Ultimate™ 3000 UHPLC system , comprised of Ultimate 3000 pump, autosampler, column compartment, and diode array detector. The signals were acquired and processed applying a Chromeleon version 6.80 software . HPLC separation was performed on a Dikma Diamonsil™ C18 column . The mobile phase was consisted of MeOH and 0.6% acetic acid in aqueous solution (v/v) in 81:19 for compound 2, 86:14 for compound 3, 70:30 for compound 4, 45:55 for compounds 5, 6, and 9, 50:50 for compound 7, with a flow rate of 1 mL/min, a column temperature of 30 °C and a injected volume of 10 μL for compounds 2–7 and 9. The detection wavelength was set at 254 nm for compounds 2 and 3, 372 nm for compound 4, 360 nm for compound 5, 276 nm for 6, 370 nm for 7, and 276 nm for compound 9. The calibration curves were constructed by plotting peak area (y) versus concentration , which concentration series (µM) were 5, 25, 50, 75, 150, 200 for compound 2; 2.5, 5, 10, 15, 25, 50 for compound 3; 5, 10, 15, 25, 50, 75 for compound 4; 2.5, 5, 10, 25, 50, 75 for compound 5; 5, 10, 25, 50, 75, 150 for compound 6; 10, 25, 50, 75, 150, 200 for compound 7; 5, 10, 25, 75, 100, 200 for compound 9. The amounts of compounds 1, 8, and 10 in the receiving chambers were below the lower limit of detection (LLOD). Quantification was carried out by peak area measurements in comparison with the calibration curves. Methodology was examined for precision, accuracy, and recovery  incubator . The integrity of the Caco-2 cell monolayer was examined by measuring the TEER with an epithelial voltohmmeter  [The human intestinal Caco-2 cell line (ATCC #HTB-37) was purchased from American Type Culture Collection . The cell culture was carried out in a Sanyo MCO-15 AC carbon dioxide  . All cel2 and 95% air at 37 °C. For confluence and differentiation, cells were seeded at a density of 1 × 105 cells/cm2 into 12-well Transwell plates  and were allowed to grow for 21 days before the permeation experiment. On day 21, the monolayers with TEER values > 500 Ω·cm2 were qualified for the transport experiment. The transport study was initiated by the careful removal of the culture medium from AP and BL sides of the inserts. Caco-2 monolayers were rinsed twice with pre-warmed HBSS and were incubated by pre-warmed HBSS for 30 min at 37 °C. Stock solutions of assayed compounds 1–10 were prepared in DMSO and diluted to 50 µM with HBSS. The final DMSO concentration was less than 2%, a concentration that did not alter the Caco-2 cells viability or permeability. The assayed solutions (50 µM) were added to the AP side  or BL side  of the inserts, while the receiving chamber contained the corresponding volume of HBSS. Incubation was performed at 37 °C for 90 min, with shaking at 50 rpm. Samples were collected from the inserts and lyophilized. The dried sample was then sonicated with the appropriate volume of MeOH, followed by centrifugation at 15,000× g for 10 min. The supernatant was filtered through a 0.45 μm filter and injected into HPLC system for quantitative analysis.The Caco-2 cell monolayer permeability was carried out according to the previously reported method ,38. BriePapp) value of each compound was calculated based on the following equation:0 is the initial test compound concentration on the donor compartment (μmol/mL), and A is the surface area of Caco-2 monolayer (cm2).The AP to BL or BL to AP permeability coefficient , 1.3 mL aliquots were taken from BL side or 0.45 mL aliquots were taken from AP side at 30 min intervals from 30 to 180 min.To observe the time-dependence, 50 μM of the compounds 2–7 and 9 were added to either AP or BL side of the inserts at the final concentration in the range of 10–175 μM for compound 2, 2.5–75 μM for compound 3, 2.5–100 μM for compound 4, 10–150 μM for compound 5, 10–200 μM for compounds 6, 7, and 9. After shaking the samples  in a shaking water bath for 90 min, aliquots were collected as described above. As in the case of time-dependent transport experiments, the kinetics curves of the compounds 2–7 and 9 were drawn at an initial concentration of 50 μM and intervals from 30 to 180 min.To observe the concentration-dependence, the compounds g for 10 min. The supernatant was filtered through a 0.45 μm filter and a 10 μL aliquot was injected into the HPLC system for analysis. To check the mass balance, the recoveries of the assayed compounds were measured at both sides of the insert and intracellular accumulation of the Caco-2 cells.To measure the amount of intracellular accumulation, the Caco-2 cells were rinsed three times with ice-cold HBSS at the end of transport experiments. After three freeze (−20 °C)-thaw (room temperature) cycles, the lysed Caco-2 cells were lyophilized. The dried cells were sonicated in 0.2 mL of 70% aqueous MeOH for 20 min and then centrifuged at 15,000× 3, 5–7, and 9, lipophilic aglycones including major triterpenoid, chalcone, and flavonone compounds in licorice, increased linearly with the concentration and saturation was not observed at the concentrations tested, indicating that the permeation mechanism for these compounds was passive diffusion. The magnitude of the bidirectional flux of these compounds was comparable to propranolol (Papp = (2.51 ± 0.21) × 10−5 cm/s). These data suggest that the compounds 3, 5–7, and 9 were well-absorbed compounds and were transported mainly through passive diffusion by the transcellular pathway. The recoveries of compounds 3, 5–7, and 9 were higher in bidirectional transport studies with very low cell accumulation, suggesting metabolism may not be involved during their intestinal absorption and transport. In spite of the fact that chalcone 4 was also an aglycone, the higher accumulation in Caco-2 cells resulted in its Papp value laying between Papp values of propranolol and atenolol, and chalcone 4 was designated as a moderately absorbed compound. Permeation of the bisglycosides 1, 8, and 10, across the Caco-2 cell monolayer was not detected, suggesting their Papp values less than 1 × 10−7 cm/s, and they were designated as the very poorly absorbed compounds. While the monoglycosides 2 with 1 × 10−6 cm/s degree of Papp value, 12 and 13 with 1 × 10−7 cm/s degree of Papp value [Papp value of different kinds of compounds may help to rich the predictive database of drug ADME/T. These new findings provided useful information for predicting their oral bioavailability, pharmacokinetics, and the clinical application as well as determination of the bioactive substance basis of Glycyrrhizae radix et rhizoma [The transport rates of the six selected compounds pp value  were des rhizoma ,40.


In particular, a specific capacitance of 1209.1 F g−1 was found for the cauliflower-like Co0.75Ni0.25(OH)2 electrode material using DTAB as the surfactant at a current density of 1 A g−1, whose structure boosted ion diffusion to present a good rate ability of 64% with a 50-fold increase in current density from 1 A g−1 to 50 A g−1. Accordingly, the asymmetric supercapacitor assembled by current LDH electrode and activated carbon electrode showed an energy density as high as 21.3 Wh kg−1 at a power density of 3625 W kg−1. The relationship between surfactant and electrochemical performance of the LDH electrode materials has been discussed.Tailoring the morphology of cobalt–nickel layered double hydroxide (LDH) electrode material was successfully achieved via the process of cathodic electrodeposition by adding different surfactants  or cetyltrimethylammonium bromide). The as-prepared Co The s2+ is a divalent ion including Ni2+, Zn2+, Mn2+; M3+ is a trivalent ion, i.e. Co3+, Al3+, Cr3+; −, Exploring electrode materials with high specific capacitance (Sc) is necessary to improve the energy density of supercapacitors. Carbon materials, conducting polymers and transition metal oxides/hydroxides with high surface area are commonly used as supercapacitor electrode materials . Carbon-2− etc.)  has neve2− etc.) . Nickel-2− etc.) .There are various techniques used to fabricate LDH electrode materials such as co-deposition , hydrothIn this paper, we proposed a facile route to synthesize cobalt–nickel LDH films of different morphologies by electrodepositing from aqueous solution in the presence of different surfactants (hexamethylenetetramine (HMT), dodecyltrimethylammonium bromide (DTAB) or cetyltrimethylammonium bromide (CTAB)). Inductively coupled plasma–optical emission spectrometry (ICP), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were applied to investigate the structures and morphologies of the as-prepared samples. Asymmetric supercapacitors (ASCs) based on current cobalt nickel LDHs as positive electrodes and activated carbon (AC) as negative electrodes were fabricated. The electrochemical performances of the ASC devices were also investigated.2.2.1.3)2·6H2O), cobalt nitrate hexahydrate (Co(NO3)2·6H2O), thiourea (CH4N2S) and HMT were acquired from China National Medicine Corporation Ltd. DTAB and CTAB were purchased from Chengdu Kelong Chemical Regent Co. Ltd (China). All chemicals were of analytical grade and used without further purification. The nickel foam was washed in 6M HCl and acetone with ultrasonication for 10 min, then washed with de-ionized water and alcohol several times, and dried in an oven at 60°C. The nickel foam substrate was rolled to a thickness of 0.2 mm before use.Nickel nitrate hexahydrate 2·6H2O, 2 mM CH4N2S with vigorous stirring, without or with 2 mM HMT, DTAB or CTAB. The samples were prepared firstly by using a multi-potential-step technique in a three-electrode configuration with Ag/AgCl as the reference electrode, platinum as the counter electrode and nickel foam as the working electrode at −2 V for 50 s under constant magnetic stirring. The obtained samples were rinsed with de-ionized water and dried at 60°C for several minutes. Then the above procedure was repeated five times. The precursors were annealed at 200°C for 2 h. The mass of active material was determined by the weight difference of the nickel foam substrate before and after deposition, and the mass loading of active material was about 0.6–1 mg per square centimetre.Electrochemical cathodic deposition was carried out for the synthesis of the LDH samples in an aqueous solution; the solution was prepared by dissolving 2.5 mM Ni(NO)2.3.α radiation λ = 0.15406 nm), SEM (JSM-7500F), TEM (JEM-2010) and X-ray photoelectron spectroscopy (XPS) (XSAM 800). The electrochemical performance was measured by electrochemical workstation (CHI660E) and LAND test system (CT2001A).The composition and structure of the samples were characterized by ICP (ARCOS SOP), FTIR spectroscopy (Nicolet 6700), XRD 2 (no surfactant), Co0.75Ni0.25(OH)2: H (HMT as surfactant), Co0.75Ni0.25(OH)2: D (DTAB as surfactant) and Co0.75Ni0.25(OH)2: C (CTAB as surfactant).0.75Ni0.25(OH)2 LDHs includes an electrochemical reaction and subsequent precipitation of mixed hydroxide, as described by the following two equations:The cathodic electrodeposition of the CoKsp) at room temperature of Co(OH)2 (2.5 × 10−16) is close to that of Ni(OH)2 (2.8 × 10−16) [× 10−16) .0.75Ni0.25(OH)2 LDHs supported on Ni foams is shown in − ions [+, CTA+) can be electrostatically adsorbed on the negatively charged surface of working electrode and expected to serve as templates [figure 1a, +, DTA+ has a shorter carbon chain length [0.75Ni0.25(OH)2: D is shorter than that (500–600 nm) of Co0.75Ni0.25(OH)2: C , which tends to aggregate to reduce the surface energy from a thermodynamic perspective [The possible formation mechanism of CoOH− ions . Meanwhiemplates . The diftructure , and reln length , so the (OH)2: C c,d, whicspective . Thus, t3.2.θ range of 10°–80°. The background peaks of nickel foam are very strong, which would disturb the diffraction peaks of electrode materials; so, cleaned stainless steel was employed to act as a substrate. As shown in θ = 11.1°, 22.2°, 38.2° are indexed as (003), (006) and (015), which originate from the characteristic lattice planes of hydrotalcite-like LDH phase [XRD analysis is a powerful tool to clarify the structure of the as-electrodeposited sample in the 2DH phase ,31. The DH phase ,33.Figu−1 and 649 cm−1, respectively [−1 is due to a small amount of intercalated sulfate [−1 is contributed by carbonate resulting from the adsorption of atmospheric CO2 and the intercalation of carbonate caused by electrodeposition process from the electrolyte dissolving CO2 in air. There is a sharp and intense band at 1384 cm−1 corresponding to the −1 and 1627 cm−1 can be assigned to stretching vibration and deformation vibration of the adsorbed water molecules as well as the interlayer water molecules [ectively . The wea sulfate . It is c sulfate . The bantructure . The banolecules . These d0.75Ni0.25(OH)2: D as a representative example was characterized by XPS analysis 2: D. The peaks can be identified as the contribution from Ni, Co, O and C, which manifest the existence of these elements in tested samples. The Co 2p spectrum (b) can be mainly split into two peaks centred at 794.9 eV for Co 2p1/2 and 779.3 eV for Co 2p3/2. Also, the spin–orbit splitting value of 15.6 eV and weak Co 2p3/2 satellite line intensity confirm the coexistence of Co2+ and Co3+ [figure 4c, the peaks located at 872.5 eV for Ni 2p1/2 and 855.2 eV for Ni 2p3/2 accompanied with two shake-up satellites are the trait of Ni2+ [figure 4d) result from O in metal–O–metal and metal–OH groups, respectively [To ascertain the chemical bonding states of every element in cobalt–nickel LDH, Coanalysis . Figure spectrum b can be and Co3+ ,38. As e of Ni2+ ,40. The ectively .Figure 0.75Ni0.25(OH)2 film obtained by electrodeposition without adding any surfactant exhibits a smooth surface with few wrinkled paper-like sheets covered on the nickel foam substrate (cf. a). The wrinkles become more pronounced and uniformly continuous on the surface of Co0.75Ni0.25(OH)2: H electrode (b). Moreover, in figure 5c for the Co0.75Ni0.25(OH)2: D sample, a single nano-sheet is grown, and the nano-sheets form a cauliflower-like 3D structure by self-assembling simultaneously. The significant 3D pores cause a loosely packed structure, which further boosts electrolyte ions to access the active materials during the fast Faradaic reaction process [0.75Ni0.25(OH)2: C sample can be found in figure 5d; the structure of a serried net-like (the inset of figure 5d) interconnected surface with inter-flake pores offers passageway for intercalation and de-intercalation of ions and electrolyte diffusion in the bulk. The TEM characterization was done by separating the Co0.75Ni0.25(OH)2: D active material from Ni foam; there are some faint nano-sheets on the grid in figure 6a–c manifesting its ultrathin nature [figure 6d) possesses broad and diffused halo, suggesting its polycrystalline feature [SEM and TEM provide further insight into the morphology and microstructure. As revealed by detailed morphologies in the images of rate cf. a. The wrlectrode b. Moreov process . The pren nature . The cor feature , which i3.3.a, it is apparent that the curves almost overlap; no significant differences of the Nyquist plots of the cobalt–nickel LDHs can be found. From the enlarged high-frequency region shown in the inset of figure 7a, by virtue of the binder-free and layered structure, no semicircles are visualized in this region, which is correlated to a low charge transfer resistance [Figure 7b exhibits the cyclic voltammetry (CV) curves of these electrodes in 2 M KOH electrolyte at a fixed scan rate of 50 mV s−1 in the potential range of 0–0.6 V. The area of the CV curve of Co0.75Ni0.25(OH)2: C is larger than that of the others, manifesting better Sc. The typical CV curves of Co0.75Ni0.25(OH)2: D at different scan rates are displayed in figure 7c, where all the curves maintain the same shape with the change in scan rates. It is associated with the improved mass transportation [The electrochemical performances of the cobalt–nickel LDHs were evaluated by a typical three-electrode experimental cell equipped with a working electrode, a platinum foil counter electrode and an Ag/AgCl reference electrode. Electrochemical impedance spectroscopy (EIS) was employed in the frequency range from 100 kHz to 0.01 Hz to understand the electrochemical reaction kinetics of the as-synthesized samples. From figure 7sistance . Figure ortation . It is eortation :3.3Ni, applied current load (A), discharging time(s) and mass of active material (g), potential window (V), respectively. The obtainable high values of Sc for Co0.75Ni0.25(OH)2: C (1318.2 F g−1), Co0.75Ni0.25(OH)2: D (1209.1 F g−1) and Co0.75Ni0.25(OH)2: H (1120 F g−1) electrodes at the current density of 1 A g−1 are in contrast to the low value for the pure Co0.75Ni0.25(OH)2 electrode (713.1 F g−1). It is thus evident that 3D structure fabricated through the use of surfactant promotes the electrochemical performance. Figure 7e displays the Scs of the cobalt–nickel LDHs synthesized with surfactants when the charge–discharge rate increases from 1 to 50 A g−1. The electrode of Co0.75Ni0.25(OH)2: D retains about 64% of the Sc at 50 A g−1, while 62% and 57% capacitance retentions for Co0.75Ni0.25(OH)2: H and Co0.75Ni0.25(OH)2: C electrodes are observed, respectively. These consequences imply that the electrodes of cobalt–nickel LDHs possess high rate capabilities. Figure 7f depicts the cycling stability tested at a current density of 8 A g−1. Seventy-five per cent of initial Sc is kept after 2000 cycles for Co0.75Ni0.25(OH)2: H, while 62% for Co0.75Ni0.25(OH)2: C and 61% for Co0.75Ni0.25(OH)2: D are retained.To further explore the electrochemical performance of the cobalt–nickel LDH electrodes, the galvanostatic charge–discharge technique was performed at the current density of 1 A gequation :3.6C=IΔt3.4.mac/mcobalt nickel LDH = 2.4 according to the following equation [+m and −m are the mass of electrode materials, c+ and c− are the specific capacitances of the positive and negative electrodes, Δ+V and Δ−V represent the potential window for the electrodes, respectively.The practicability of these materials was investigated by assembling ASCs using the cobalt–nickel LDHs as positive electrodes and AC as negative electrodes in 6M KOH aqueous electrolyte. It is found that the optimal mass ratio to fabricate the ASC device is equation :3.7m+m−=a, all EIS plots of these ASCs contain a semicircle in the high-frequency region and then a straight sloping line; the smallest diameter of the semicircle of Co0.75Ni0.25(OH)2: D//AC is associated with a smallest charge transfer resistance (RCT) [RCT is pretty vital to high electrical conductivity and outstanding rate capability [figure 8b were obtained in the window of 0–1.45 V at a scan rate of 50 mV s−1. The CV curves of the Co0.75Ni0.25(OH)2: D//AC keep the same shape at various scan rates, which means the fast charge–discharge property of the device (figure 8e). From the galvanostatic charge–discharge curves at the current density of 0.2 A g−1 (figure 8c), the discharge time of Co0.75Ni0.25(OH)2//AC is shortest among these ASCs. Energy density (E) and power density (P) were estimated using the following equations [As can be seen from figure 8ce (RCT) . The lowpability . Benefitquations :3.8E=12C−1 (Co0.75Ni0.25(OH)2: C//AC), 28.4 Wh kg−1 (Co0.75Ni0.25(OH)2: D//AC) and 26.7 Wh kg−1 (Co0.75Ni0.25(OH)2: H//AC). This result further implies that the use of surfactant during the electrodeposition process is advantageous to the electrochemical performance. As the current density increases from 0.2 to 5 A g−1 shown in figure 8f, the calculated Sc value of the Co0.75Ni0.25(OH)2: D//AC ASC device decreases from 97.3 to 73.1 F g−1. As shown in figure 8d, the trend in rate capabilities of Co0.75Ni0.25(OH)2: D//AC (75%), Co0.75Ni0.25(OH)2: H//AC (69.3%), Co0.75Ni0.25(OH)2//AC (65%) and Co0.75Ni0.25(OH)2: C//AC (54.8%) is similar to the trend in the diameters of the semicircles in the inset of figure 8a. This observation supports the fact that Co0.75Ni0.25(OH)2: D//AC has better interfacial faradic reaction kinetics; the Co0.75Ni0.25(OH)2: D//AC device also keeps a better cycle life (65%) after 1000 cycles in figure 8h. The two important results are owing to the preferable electronic conductivity and unique surface structure; the cauliflower-like structure of Co0.75Ni0.25(OH)2: D has many interspaces between the particles, which allow the volume expansion to take place easily, and promote the high distribution of active sites as well as high speed pathway for continuous electronic and ionic transportation [figure 8g. A maximum power density (3625 W kg−1) at an energy density of 21.3 Wh kg−1 was achieved by the Co0.75Ni0.25(OH)2: D//AC ASC device, which is in line with the conclusion that a high rate ability of the electrode material favours supercapacitor providing a high power density [0.75Ni0.25(OH)2: D//AC electrode does not present the highest Sc, it still seems more suitable for practical application on grounds of its preferable rate capability and cycle life.The energy densities of the devices can be further calculated to be 28.6 Wh kgortation . Ragone  density . Althoug4.0.75Ni0.25(OH)2 active material having a Sc of 713.1 F g−1, the modified Co0.75Ni0.25(OH)2 LDHs with distinct surface morphologies showed superior Scs: wrinkle-like, cauliflower-like and net-like Co0.75Ni0.25(OH)2 electrodes exhibited Sc of 1120 F g−1, 1209.1 F g−1 and 1318.2 F g−1, respectively. Among ASC devices fabricated using Co0.75Ni0.25(OH)2 LDHs as positive electrodes, the Co0.75Ni0.25(OH)2: D//AC ASC device showed a preferable energy density at the same power density. These results demonstrated that morphology modification by surfactant enhanced the electrochemical performance in cobalt–nickel LDH electrode materials.The morphologies of cobalt–nickel LDHs were successfully modified by using surfactants during the process of cathodic electrodeposition. In comparison to the pristine Co


Understanding the deactivation mechanism of 2-deoxy-d-ribose-5-phosphate aldolase by its natural substrate leads to a single mutant showing complete acetaldehyde resistance. d-ribose-5-phosphate aldolase (DERA) is used in organic synthesis for the enantioselective reaction between acetaldehyde and a broad range of other aldehydes as acceptor molecules. Nevertheless, its application is hampered by a poor tolerance towards high concentrations of acetaldehyde, its natural substrate. While numerous studies have been performed searching for new, more acetaldehyde-resistant DERAs, the mechanism underlying this deactivation process has remained elusive. By using NMR spectroscopy on both the protein and the small-molecule scale, we could show that a reaction product binds to the inner part of the enzyme, and that this effect can be partly reversed via heating. The crystal structure of DERA before and after acetaldehyde incubation was determined at high resolution, revealing a covalently bound reaction product bridging the catalytically active lysine (K167) to a nearby cysteine (C47) in the deactivated enzyme. A reaction mechanism is proposed where crotonaldehyde as the aldol product of two acetaldehyde molecules after water elimination forms a Schiff base with the lysine side chain, followed by Michael addition of the cysteine thiol group to the Cβ atom of the inhibitor. In support of this mechanism, direct incubation of DERA with crotonaldehyde results in a more than 100-fold stronger inhibition, compared to acetaldehyde, whereas mutation of C47 gives rise to a fully acetaldehyde-resistant DERA. Thus this variant appears perfectly suited for synthetic applications. A similar diagnostic and preventive strategy should be applicable to other biocatalysts suffering from mechanism-based inhibition by a reactive substrate, a condition that may be more common than currently appreciated in biotechnology.2-Deoxy- In this study we focus on two main questions: (1) by which mechanism does acetaldehyde deactivate the enzyme? (2) Is there a way to prevent/withdraw this effect? Until now there has only been one study by Jennewein et al.However, practical application of DERA in organic synthesis is limited by its poor stability towards high acetaldehyde concentrations.Ka of the lysine sidechain – resulting in an uncharged residue at neutral pHvia mass spectrometry after incubation of DERA with acetaldehyde and a subsequent reducing step with sodium borohydride revealed an ethylation pattern in 14 of 16 outer lysines. Thus it could be indirectly shown that acetaldehyde forms Schiff bases with the lysines on the protein surface. To find out whether there is a relationship between the number of outer lysines and the stability of DERA towards high acetaldehyde concentrations, the genome database was searched for DERA genes coding for a protein with fewer lysine residues. The orthologue from Corynebacterium bovis was found to expose only a single surface lysine. However, this aldolase did not show increased stability towards acetaldehyde, compared to DERAEC , with an overall root-mean-square distance of 0.38 Å for all resolved main-chain atoms. This observation supports the utility of the double mutant for structural investigations. A -TROSY spectrum of the monomeric DERA was recorded before and after acetaldehyde incubation  was used that has virtually identical biochemical properties.cubation . Most siU-13C,15N]-DERA monomer using HNCA, HN(CO)CA, HNCO and HN(CA)CO experiments. Information about Cβ atoms of the sidechains could not be obtained via HNCACB and HN(CO)CACB triple resonance experiments due to a low signal-to-noise ratio (probably related to the size of the protein).1H,1H,15N]-TOCSY-HSQC experiment was performed, where magnetization is transferred between the 1H spins of the sidechains and then via the 15N nuclei to the HN-protons for detection.β and Hα nuclei of the alanine residues. With this information at hand it was possible to assign all amino acids except the terminal segments and S239, which is located in a loop region . The assigned TROSY spectrum is shown as ESI . For quality control the secondary structure of the protein was determined with CSI 2.0 (1JCL). The results revealed a high correlation between experimental and calculated values and are available as ESI , [1H–1H]-TOCSY and [1H–1H]-COSY experiments. The assignment was supported by a recently published NMR study on acetaldehyde in aqueous solution.DERA catalyzes a sequential aldol reaction, resulting in addition products formed out of two or three acetaldehyde molecules. The second product is converted into a lactol by spontaneous cyclization.U-13C]-acetaldehyde, and after several extensive washing steps a 13C-NMR spectrum of the enzyme was recorded  indicate that there are still free aldol products in solution (probably caused by an equilibrium between a protein-bound and an unbound state), some broad signals appear at 55 and 42 ppm, suggesting association with the protein. The whole spectrum is given as ESI . In addition, new signals appeared. In some cases two states reflecting the situation before and after acetaldehyde treatment, respectively, appeared to coexist. The spectrum can be interpreted as an equilibrium or mixture between the active and inactive DERA variants, which is in agreement with the kinetic measurements of the monomeric aldolase showing 50% of the initial activity after heating. Subsequently, the 13C spectrum of DERA incubated with 13C-labeled substrate was recorded again after the heating step. In 13C-labeled molecules from the protein. As expected, the DOSY spectrum  of the DERA catalyzed reaction appear to interact with the protein and may thus block the binding pocket prompted us to search for strategies to overcome this deactivation. Specifically, we tested if enzyme activity could be recovered by means of heating, as the dimeric DERA is rather thermostable and remains fully active after several hours of incubation at 55 °C (50 °C in case of the monomeric variant).E. coli before and after acetaldehyde incubation as well as after thermal treatment have been determined (From our NMR studies we conclude that product(s) derived from the DERA-catalyzed reaction bind to the inner part of the protein, resulting in an inactive enzyme. To determine the structure of these reaction product(s) and to identify the exact target site(s) within the protein, X-ray structures of DERA from termined . Crystal2 in All three structures turned out to be very similar, with obvious alterations limited to the catalytic center. Interestingly, the native enzyme features electron density for a six-atom molecule with about 70% occupancy, which is attached to the active lysine residue . The covSubsequent heating at 50 °C results in further  changes in the crystal structure. Specifically, we observe a reduction in occupancy of the bridging ligand , which is accompanied by the appearance of a weak difference density possibly indicating partial displacement of the inhibitor (not shown). Together, these observations strongly suggest that acetaldehyde at high concentrations or, more specifically, the formed homoaldol adduct acts as a mechanism-based inhibitor of DERA. At increased temperatures, the peculiar thioether link undergoes time-dependent elimination, accounting for gradual recovery of enzyme activity.β atom is missing, and second, the substrate has formed a covalent bond to C47. Our NMR studies have demonstrated that 3-hydroxybutanal is prone to water elimination, resulting in loss of the hydroxyl group and formation of the respective α,β-unsaturated aldehyde (see 2 and 6). As shown in 2 hybridized Cβ atom of crotonaldehyde. Indeed, incubation of DERA with this particular compound yielded more than 100-fold stronger inhibition, compared to acetaldehyde; specifically, a concentration as low as 1 mM leads to virtually full inactivation within less than one hour -2-pentenal. The initial idea was to determine the orientation of the molecule linking C47 and K167 based on the additional carbon atom, compared to crotonaldehyde [(E)-2-butenal]. However, the larger compound turned out not to form a covalent bond with C47. Our crystal structures indeed suggest that the bulky ethyl substituent cannot be easily accommodated by the pocket lined by D16, T18, C47, and K201, which may prevent Michael addition of the thiol to the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd2IvFj" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>C bond. Notably, such steric hindrance would not appear relevant for the alternative orientation, where the ethyl moiety would be facing the substrate channel.It should be noted that, based on electron density alone, we cannot strictly exclude a reverse orientation of the covalently bound ligand. In an alternative reaction mechanism, the nitrogen atom of K167 might act as a nucleophile for Michael addition to crotonaldehyde, while the cysteine residue could form a hemithioacetal with the aldehyde group  showed a frequency of 91% for cysteine at position 47. Thus, although being highly conserved, C47 is not essential for catalytic activity.5Since the catalytic K167 cannot be exchanged without losing the enzyme function, a viable strategy to prevent this irreversible deactivation might be mutation of the C47 residue. A sequence alignment of The critical cysteine has been replaced by methionine, a rather similar but non-nucleophilic amino acid, by means of site-directed mutagenesis. Biochemical characterization of the new variant showed that it retains 60 ± 5% of the wildtype activity towards its natural substrate, and the melting temperature of 65.1 °C suggest a similar thermostability as found for wildtype DERA (66.6 °C). Both proteins were incubated with different concentrations of acetaldehyde and the activity was recorded as a function of time.As can be seen in i.e., a single point mutation, we were able to create a fully acetaldehyde resistant E. coli DERA.These results provide further support for the inactivation mechanism depicted in E)-2-butenal], which is formed upon water elimination from the aldol product between two acetaldehyde molecules.It is interesting to note that already in 1976, a long time before any structural information on this enzyme was available, D. C. Wilton identified acrolein  as an irreversible inhibitor of DERA and proposed an inactivation mechanism where first a Schiff base is formed between the aldehyde and the catalytic lysine and subsequently an unknown nucleophile forms a covalent bond with the Michael system.To test the potential of the new mutant in organic synthesis, reaction kinetics of both wt and C47M DERA were assessed by NMR spectroscopy in 300 mM and 1 M acetaldehyde. As can be seen in To validate if this new mutant is still as versatile a catalyst as the wildtype DERA, we performed a  HPLC-MS analysis of DERA-catalyzed reactions using acetaldehyde as donor and various acceptor aldehydes, which revealed the same pattern of the respective single and double aldol products as found with wildtype DERA . Indeed E. coli (see Introduction). However, a sequence alignment revealed the same critical cysteine at position 47 – except for one example of Hyperthermus butylicus (unknown structure)E. coli DERA suggest a relationship between the half-life of that enzyme in acetaldehyde and the amount of protein used in the assay 32Covalently binding inhibitors, often effecting mechanism-based (or suicide) inhibition, have evolved as an important class of compounds regulating enzyme activity; a particularly well-understood case is the superfamily of serpins, which are involved in downregulation of proteases.et al. analyzed the role of acetaldehyde as the product of the lipase-catalyzed reaction between vinyl esters and alcohols. Using mass spectrometry they were able to identify aldol condensation products such as crotonaldehyde, which form Michael adducts with surface-exposed amino acids.S-nitroglutathione by the human carbonyl reductases 1 and 3.On the other hand, mechanism-based inhibition as an unwanted side reaction in biocatalysis is rarely described in literature. While studies on optimizing biocatalysts towards high operating temperature or stability in organic solvents are published regularlyIn this study, we have identified crotonaldehyde, the product of aldol condensation between two acetaldehyde molecules, as a mechanism-based inhibitor of DERA. This substrate acts by covalently linking the catalytic lysine residue to a nearby cysteine. To our knowledge, this is the first detailed account of mechanism-based inhibition by a reaction product derived from a natural substrate in biocatalysis. Ultimately, these findings are related to the more general challenge of using this reactive substrate  in synthesis, involving many side reactions and possible binding of the products to the enzyme. In the case of DERA, an elegant solution is achieved by replacing a single reactive residue, yielding an acetaldehyde-resistant enzyme. We are confident that our results will inspire similar efforts to improve the performance of enzymes in biotechnology applications.All chemicals and enzymes, if not mentioned separately, were purchased at Carl Roth (Karlsruhe), Sigma Aldrich (Steinheim) or (for labeled compounds) at the Cambridge Isotope Laboratories (Saarbrücken) in analytical grade.deoC-gene from C. bovis (codon harmonized) was synthesized by GenScript and cloned into the pET21a-vector via NdeI and EcoRI restriction sites. The deoC-gene from E. coli had been isolated previously.via round-the-hornE. coli BL21(DE3) strain in TB-medium. For 15N- and/or 15N–13C-labeled proteins M9 minimal media according to Sivashanmugam et al.g).DERA-coding i)-buffer at pH 7.0, disrupted via sonification, and after centrifugation (15 min at 12 000 × g) the supernatant was loaded onto a NiNTA-column in a cyclic process (15 min). After washing with KPi-buffer containing 30 mM imidazole the enzyme was eluted with 250 mM imidazole. PD-10 desalting columns were used to rebuffer the solution to 20 mM KPi, and purity (≥95%) was verified with SDS-PAGE. The protein concentration was determined by the Bradford assay.20For enzyme purification 1–4 g cells were resuspended in 5 volumes of 20 mM potassium phosphate . 13C-labeled reaction products were separated from the protein using a centrifugal filter device (VivaSpin). For NMR kinetics, a solution of 2 mg mL–1 protein in 120 mM KPi pH 7 including 5% D2O was prepared. Directly before measurement a solution of acetaldehyde in 100 mM KPi pH 7 was added, resulting in a final concentration of 300 mM and 1 M, respectively. The measurements were performed over 2 h.For NMR studies the pH of the solution was adjusted to 6.8, the protein was concentrated to 0.2–0.4 mM and 5% Dvia size exclusion chromatography was performed. A SuperdexTM200 10/300 GL  column was equilibrated in 100 mM KPi, 150 mM NaCl (pH 7); 5 mg protein was loaded on the column, and the elution profile was recorded by means of absorbance at 280 nm.Prior to crystallization screening a second purification step d-ribose-5-phosphate (DRP) was measured for 1 min at 25 °C. In a retro-aldol reaction, glyceraldeyhde-3-phosphate is formed. The latter is reduced to glycerol-3-phosphate by the auxiliary enzymes glycerol-3-phosphate dehydrogenase (GDH) and triose phosphate isomerase (TPI) under NADH consumption, which was detected with a photometer at 340 nm.–1); the first kinetic measurement was performed immediately after mixing. Additional samples were taken at regular intervals and their activities determined. Note that the samples contained both enzyme and acetaldehyde used for pre-incubation. This aldehyde may function as product inhibitor for the retro-aldol reaction which is exploited for activity measurement. As a consequence, product inhibition will become the weaker the more acetaldehyde is converted to an aldol product by an active DERA, possibly explaining the time-dependent increase in activity of the C47M mutant.The activity for cleavage of the natural substrate 2-deoxy-EC: 2.2 mg with 8.3 U mg–1, C47M mutant: 4.4 mg with 5 U mg–1), dissolved in 0.5 mL of triethanolamine buffer . Each acceptor aldehyde was used with equal substance amount (0.10 mmol). The extracted and dried samples were dissolved in acetonitrile. HPLC-MS measurements were performed on an Agilent1100 HPLC system equipped with a C18 Eclipse Plus column  interfaced with a Q-TRAP 4000 (ABSciex). As solvent a mixture of (A) H2O with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid was used, applying a flow rate of 0.8 mL min–1 and the following gradient: 60% A – 2 min, then 10% A – 25 min. The particular product mass traces were extracted.The aldol reactions were performed on an analytical scale according to a protocol established in our laboratory.–1) was incubated for 3 h with 300 mM acetaldehyde in 200 mM sodium borate buffer, pH 8.5, followed by reduction in sodium borohydride (400 mM) to stabilize the Schiff bases.–1 trypsin) the protein sample was desalted by RP C4 ZipTips (Millipore) and analyzed by LC-MALDI-TOF/TOF mass spectrometry .DERA (6 mg mLZ-axis pulse-field-gradient (PFG) triple resonance probes. For 1H and 13C chemical shifts 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as internal standard at 0 ppm. 15N chemical shifts were referenced indirectly using frequency ratios.1H measurements were recorded relative to the resonance of the solvent. Assignment of protein backbone resonances was performed using a combination of multidimensional NMR experiments: 2D [1H–15N]-TROSY,1H–1H–15N]-TOCSY-HSQC51NMR experiments were conducted at 25 °C on Bruker Avance III HD (600 and 700 MHz), Bruker Avance/DRX 600 (600 MHz) and Varian VNMRS (900 MHz) instruments, equipped with cryogenic E. coli DERA (K58E-Y96W mutant) were subjected to crystallization screening at 20 °C in a sitting-drop setup, using a robotic system . Crystals were observed for a number of conditions, including reservoir solutions containing 10% (w/v) PEG4000, 20% (v/v) 2-propanol (native enzyme), or 20% (w/v) PEG4000, 10% (v/v) 2-propanol, 0.1 M HEPES pH 7.5 , and protein concentrations of 10–20 mg mL–1. Prior to cryocooling, crystals were soaked in reservoir buffer containing 7–10% (v/v) glycerol. Single-wavelength diffraction datasets were recorded at 100 K on beamlines ID23-1 and ID29 of the European Synchrotron Radiation Facility  equipped with PILATUS 6M and PILATUS3 6M detectors (Dectris), respectively. Data processing was carried out using XDS and XSCALE.52Samples of monomeric E. coli DERA belonged to space group C2. Initial phases for the native enzyme were obtained by molecular replacement with MOLREP,1JCL) as template. The crystals contain a single chain per asymmetric unit, corresponding to a Matthews coefficient of 1.9 Å3 Da–1 and a solvent content of 37%. The initial model was improved iteratively by alternating reciprocal space refinement in phenix.refine5EKY (native monomerized DERA), ; 5EL1  and ; 5EMU .Crystals of monomeric Ribbon representations in 59M. D., T. C., P. N. and J. P. conceived the project; M. D. cloned, purified and characterized the enzymes; R. H. and M. D. performed the NMR studies with assistance from M. S. for the backbone-assignment. C. B. with support from R. H. and M. D. measured NMR kinetics; O. H. W. with assistance from M. D. and T. C. crystallized the enzymes and determined the structures; C. B. performed the biotransformations; the manuscript was drafted by M. D. and finalized with contributions from all authors; D. W. and J. P. supervised research.Supplementary informationClick here for additional data file.


We aimed to figure out the molecular network of PVT1 and EZH2 on hepatocellular carcinoma (HCC) cells growth. We also explored the interaction between PVT1, EZH2, MDM2 and P53.Microarray analysis was performed to screen for abnormally expressed genes in HCC tissues and PVT1 was identified as one gene significantly upregulated in HCC. CCK-8 assay, colony formation assay, and flow cytometry detected cell vitality, proliferation and apoptosis, respectively. RIP and RNA pull-down assays were employed to examine the connection between PVT1 and EZH2. The effect of PVT1 on the stability of EZH2 protein and the impact of EZH2 on MDM2 were detected by ELISA. Co-immunoprecipitation assay was used to evaluate the relationship between MDM2 and EZH2. Western blot detected the expression of EZH2, MDM2 and P53.EZH2 mRNA expression. EZH2 protein stabilized MDM2 protein expression by binding to MDM2 protein. PVT1 enhanced the protein expression of EZH2 and MDM2 as well as inhibited P53 protein expression.Up-regulated PVT1 was detected in HCC. Knockdown of PVT1 inhibited HCC cell propagation and promoted apoptotic cells. PVT1 could improve EZH2 protein stability by binding to EZH2 protein but have no significant impact on PVT1 promoted HCC cell propagation and inhibited apoptotic cells by recruiting EZH2, stabilizing MDM2 protein expression and restraining P53 expression.The online version of this article (10.1186/s12935-018-0582-3) contains supplementary material, which is available to authorized users. As an aggressive malignancies, hepatocellular carcinoma (HCC) results in high mortality of patients . DespiteLong non-coding RNAs (lncRNAs) with more than 200 nucleotides are short of protein coding potential . LncRNAsThe plasmacytoma variant translocation 1 gene (PVT1) is a copy number amplification-associated lncRNA . PVT1 fuEZH2) is a subunit of the multi-enzyme complex polycomb repressive complex 2 and is involved in chromatin compaction and gene repression [EZH2 was verified to contribute to the aggressiveness of various human cancers [EZH2 promotes cell proliferation in laryngeal carcinoma [EZH2 in HCC has been found in some studies. For instance, up-regulated EZH2 was measured in HCC tissues, which was positively correlated with tumor grade and clinical stage [EZH2 inhibited HCC cell growth through inhibition of β-catenin signaling [EZH2. For example, PVT1 was verified to induce the increase of EZH2 in gastric cancer [EZH2 in HCC remains unclear.The enhancer of zeste homolog 2 , using the Human 8 × 60 k LncRNA Expression Microarray V3.0 . Total RNA was quantified with NanoDrop 2000 . After synthesized with total RNA, cDNA was labeled according to Nimblegen Gene Expression Analysis protocol . Data analysis was conducted by NimbleScan .Three Human HCC cells  and a normal liver cell (HL-7702) were purchased from the BeNa Culture Collection . HepG2 and SK-HEP-1 in minimum essential medium  were cultured with 10% fetal bovine serum ; Huh7 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS (GIBCO BRL); BEL-7404 and HL-7702 were maintained in RPMI1640 medium  supplemented with 10% FBS (GIBCO BRL).−∆∆Ct method determined comparative quantification. Primer sequences were exhibited at Table TRIzol Reagent  and NanoDrop 2000  were utilized to isolate and quantify total RNA. RNA was reversely transcribed to cDNA by using ReverTra Ace qPCR RT Kit . QRT-PCR was conducted by THUNDERBIRD SYBR qPCR Mix . Internal controls were U6 and GADPH. 26). Transfection was performed by Lipofectamine 2000 . Cells transfected with pcDNA3.1 served as NC group; cells transfected with PVT1-pcDNA3.1 was regarded as PVT1 group; cells transfected with si-PVT1 and pcDNA3.1 served as si-PVT1 group.Si-PVT1, PVT1-pcDNA3.1 and pcDNA3.1 were synthesized by GenePharma . Cells were seeded into 6-well plates  at 24, 48, 72 and 96 h according to the manual. Absorbance was recorded at 490 nm.8 × 103/well) for 2 weeks. After discarding the medium, we used 4% paraformaldehyde to fix cells for 15 min, and added Giemsa solution to stain for 5 min.Cells transfected with si-NC or si-PVT1 were collected at logarithmic growth phase. Afterwards, cells were placed in a 6-well plate . Cells were re-suspended in 500 μL binding buffer containing 5 μL propidium iodide (PI) and 5 μL annexin V-FITC. Cells were analyzed by BD Accuri C6 flow cytometer .EZH2, ab191080, 1:500; anti-MDM2, ab38618, 1:1000, Abcam, Cambridge, MA, USA). After washed three times with TBST, second anti-body was added  for 1.5 h. Signal detection was carried out with an ECL system .Protein samples were harvested from cells to were lysed in RIPA buffer . The concentration of protein was determined using Pierce BCA Protein Assay Kit . After separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to polyvinylidene difluoride membranes , blocked with TBST including 5% non-fat skimmed milk. Primary antibodies were added  according to the manual. The supernatants of cell extracts were incubated with treated-beads for 6 h. We used the RIP wash buffer to wash the beads for 6 times. The purified RNA was used for qRT-PCR analysis.PVT1 and the antisense RNA were transcribed by mMESSAGE mMACHINE T7 Kit  and RNeasy Mini Kit  in vitro, biotin-labeled using Pierce RNA 3′ End Desthiobiotinylation Kit . 1 mg total protein from HepG2 to Huh7 cell extracts were mixed with 50 pmol of biotinylated PVT1 for 1 h, and then added with 60 µL of Streptavidin Beads (Invitrogen) for 1 h. The proteins were resolved by 10% SDS-PAGE, detected by conventional western blot analysis.EZH2, HepG2 and Huh7 cells in 24-well plates (2 × 105/well) were added with cyclohexane . Total protein was extracted from cell culture media using RIPA buffer , followed by blocking with 5% non-fat PBS for 1 h. After participation of primary antibody for 30 min, cells were added with second antibody for another 30 min and then added with TMB for 15 min. 2 mol/L H2SO4 was added for 5 min and the optical density was read at 450 nm using a VICTOR3 Microplate Reader .After transfected with si-NC, si-PVT1 or si-For co-immunoprecipitation, HepG2 and Huh7 cells at 48-hour post-transfection were washed twice with PBS and lysed in CytoBuster Protein Extraction Reagent (Millipore). Cell lysates were purified with Protein G Plus-Agarose beads (Santa Cruz) and incubated with prepared antibody-conjugated beads overnight. The beads were dissolved in 30 μL SDS-PAGE loading buffer for protein analysis with the indicated antibodies.t test and Chi square tests. A probability level of 0.05 was chosen for statistical significance.GraphPad Prism 6.0 software  analyzed all statistics. The measured parameters presented as mean ± SD. Statistical tests for data analysis were two-tailed P < 0.05). The results of qRT-PCR confirmed overexpression of PVT1 in HCC tissues . CCK-8 and colony formation assays elucidated that cell vitality and proliferation in si-PVT1 group were attenuated compared with control group . Furthermore, knockdown of PVT1 induced apoptosis in HepG2 and Huh7 cells through flow cytometry analysis .QRT-PCR evaluated PVT1 expression in different HCC cell lines. As shown in Fig. EZH2-RNA participation compared with si-NC group . In addition, knockdown of PVT1 had no impact on EZH2 mRNA expression . However, PVT1 was not directly bound to MDM2 or P53 . Western blot and ELISA assays demonstrated that si-PVT1 decreased the EZH2 protein stability . Taken together, PVT1 could improve EZH2 protein stability but have no significant impact on EZH2 mRNA expression.RIP experiments were conducted in HepG2 and Huh7 cells, which revealed PVT1 enrichment in ion Fig. b and RNAEZH2 and si-MDM2 . Measured by western blot, knockdown of EZH2 decreased MDM2 protein expression, whereas knockdown of MDM2 had no significant impact on EZH2 protein expression . Therefore, EZH2 protein improved the stability of MDM2 protein.In Co-IP assay, we found that EZH2 could bind to MDM2 Fig. a. To furion Fig. c. MeanwhAfter transfected with pcDNA3.1-PVT1 and si-PVT1 in HepG2 and Huh7 cells, western blot was employed to determine the protein expression of EZH2, MDM2 and P53. The results elucidated that overexpression of PVT1 improved the protein expression of EZH2 and MDM2 as well as inhibited P53 protein expression. In contrast, knockdown of PVT1 had the opposite effects  RNA pull-down assay indicated that PVT1 could not directly bind to MDM2 or P53.


Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host Trypanosoma brucei and, coupled with deletion experiments, demonstrates that histone variants act as a molecular link between genome architecture, chromatin conformation and antigen variation.Long-read sequencing allows the assembly of antigen-gene arrays in  These assemblies suggest that the linear organization of the genome may be important for restricting high levels of recombination to regions that code for antigens and for ensuring that all but one antigen is repressed.Genome sequences of several pathogens have revealed a partitioning of chromosomes, with housekeeping genes often being located in the central core and antigen genes being located in subtelomeric regions4, which has highlighted the critical role of spatial organization and compartmentalization of DNA in the regulation of gene expression and recombination3. In addition, microscopy-based analyses of the unicellular eukaryotic parasites Plasmodium falciparum and T. brucei have indicated that nuclear organization may be important for the mutually exclusive expression of antigens9. However, to our knowledge, the proteins that are involved in shaping genome architecture and controlling antigen expression have not yet been identified in any organism.Recently, genome-wide Hi-C analyses have begun to uncover the 3D organization of chromosomes at high resolutionThis study aimed to identify the process that restricts antigen expression. Specifically, we sought to identify proteins that are important for maintaining genome architecture and to determine whether global and/or local changes in chromatin conformation affect antigen expression.T. brucei—which is the causative agent of human sleeping sickness—the key antigens are the variant surface glycoproteins (VSGs). Most VSG genes—of which there are about 2,500—are found in long subtelomeric arrays of megabase chromosomes6. In addition, about 65 VSG genes are located on mini-chromosomes (50–150 kb in length)10 and a smaller subset of VSG genes is located in distinct telomere-proximal polycistronic transcription units, called expression sites11. Expression sites are grouped into metacyclic-form and bloodstream-form expression sites  on the basis of the life-cycle stage during which they can be activated. VSG genes are transcribed only when they are located within an expression site and only one of about 15 BESs is transcribed at a time, which ensures that the expression of VSG genes is mutually exclusive11. Therefore, a genome sequence that contains both subtelomeric VSG gene arrays and telomeric expression sites, which is lacking in the available T. brucei genome (isolate TREU 927)6, is required to elucidate the molecular link between genome architecture and antigenic variation.In T. brucei 427 Lister isolate (the most commonly used laboratory isolate) and assembled the reads into megabase chromosomes, of which there are 11  sequencing technology, we generated an approximately 100-fold genome-sequence coverage of the T. brucei genome: centromeres and junctions between the core and subtelomeres function as the most prominent boundaries of DNA compartments. In addition, the frequency of DNA–DNA contact was substantially higher across subtelomeric regions compared to core regions, which indicates that subtelomeres are more compact than the core region  is present in T. brucei and the depletion of this subunit causes deregulation of VSG expression17. However, it has remained unclear whether this is a direct effect because SCC1 depletion strongly affects cell-cycle progression and growth rate, leading to rapid parasite death18.Higher-order genome structures are established and maintained by architectural proteins such as CCCTC-binding factor (CTCF) and cohesinT. brucei SCC1 is enriched across tRNA and rRNA genes, termination sites of RNA polymerase II transcription and most of the 3′ ends of BESs . This raised the possibility that these two histone variants function together with SCC1 in shaping genome organization and the regulation of antigen expression.Chromatin immunoprecipitation with sequencing (ChIP–seq) revealed that in ESs Fig. . This paH3.V, ΔH4.V and ΔH3.VΔH4.V cells. No cell cycle defect was observed in these cell lines , and homogenously express VSG-2 . Thus, an increase in heterogeneity of VSG gene expression can be caused by a loss of mutually exclusive expression of VSG genes in individual cells—that is, heterogeneity in antigen expression at the single-cell level—or an increased switching frequency in expression of VSG genes in different parasites (heterogeneity at the population level).Laboratory-adapted isolates, such as the one used here, switch their expression of VSG isoforms at very low frequency  of individual nes Fig. , 7. Actiame Fig. , the perT. brucei, the switching of expression of VSG genes occurs by two distinct mechanisms11: either by switching transcription from one BES to another  or by a recombination-based event that leads to the replacement of the previously active VSG gene with a new VSG gene from a different genomic location  gene pair that was present in both BES1 and BES15. The data also revealed that the new chimeric BES contained three copies of ESAG8, one from BES1 and two from BES15 . Hi-C data from ΔH3.V cells revealed marked changes in inter-chromosomal interactions  at 37 °C up to a density of 106 cells/ml. If required, drugs were used at standard concentrations.All H3.V and ΔH4.V cells used in this study have previously been published32. After generation of a transgenic cell line, the correct tagging of a gene or the deletion of gene was verified by PCR. Cell lines were not tested for mycoplasma contamination.Δcells. To delete the first H3.V allele (Tb927.10.15350), the regions upstream and downstream of the H3.V CDS were PCR-amplified using the following primer pairs: H3.V_01_F, H3.V_02_R and H3.V_03_F, H3.V_04_R (see Supplementary Table 33 using InFusion HD Cloning Plus reagents (Clontech) at PvuII/HindIII and BamHI/SbfI restriction sites. The resulting plasmid was linearized with PvuII and SbfI and stably transfected into the H3.V locus of T. brucei wild-type cells to generate H3.V/ΔH3.V cells. To add an N-terminal 2× Ty1 tag to the second H3.V allele, the sequence of 326-bp upstream of the H3.V CDS  was cloned into the ApaI/NotI linearized vector pPOTv3-2×Ty1 using InFusion HD Cloning Plus reagents (Clontech). Downstream of the blasticidin resistance marker and the Ty1-tag, a 417-bp DNA sequence homologous to the H3.V CDS 5′-end  was amplified (leaving out the ATG start codon) and likewise inserted using SacI and NheI restriction sites. The tag sequence was subsequently replaced by a codon-optimized version: oligonucleotides containing two codon-optimized Ty1 coding sequences (H3.V_09 and H3.V_10) were annealed, digested with HindIII and SacI and ligated into the HindII/SacI-linearized plasmid. Finally, the plasmid was linearized with ApaI and NheI restriction enzymes and stably transfected into H3.V/ΔH3.V cells to generate Ty1-H3.V/ΔH3.V.Ty1-H3.V/ΔH3.V double-knockout cells. To delete H4.V, the upstream  and downstream  regions flanking the H4.V CDS (Tb927.2.2670) were amplified from bloodstream-form wild-type gDNA and purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). The PCR product of the upstream region was inserted into the plasmid pyrFEKO-Neo33 using InFusion HD Cloning Plus reagents (Clontech) between HindIII and AgeI restriction sites. The downstream region was integrated by ligation using BamHI and SbfI restriction sites. The neomycin resistance cassette was exchanged with a blasticidin or a phleomycin resistance marker, respectively, using the BglII and XbaI sites that flank the resistance marker. To this end, the blasticidin and phleomycin cassettes were excised from pyrFEKO-BSD or pyrFEKO-Phleo, purified and ligated into the target plasmid. The plasmids were linearized with NheI and SbfI and stably transfected into the previously published ΔH3.V cell line32.ΔH3.VΔH4.V cells. To delete the first SCC1 allele (Tb927.7.6900), the flanking regions upstream  and downstream  of the SCC1 CDS were amplified, digested with PvuII/HindIII and BamHI/SbfI, respectively, and ligated into pyrFEKO-Hyg33 at PvuII/HindIII and BamHI/SbfI restriction sites. Wild-type cells were transfected with the linearized plasmid (PvuII/SbfI) to obtain SCC1/ΔSCC1 cells. For the N-terminal Ty1-tagging of the second SCC1 allele, the 3′ end of the SCC1 5′ UTR was amplified , digested with ApaI and NotI and ligated into the ApaI/NotI-linearized vector pPOTv3-2×Ty1. Next, the 5′ end of the SCC1 CDS was amplified (leaving out the ATG start codon) , digested with SacI and NheI and ligated into the likewise-digested plasmid. The Ty1-tag was exchanged by a codon-optimized version as described for N-terminal tagging of H3.V (see above). The ApaI/NheI linearized plasmid was stably transfected into SCC1/ΔSCC1 T. brucei cells to generate Ty1-SCC1/ΔSCC1.Ty1-SCC1/ΔSCC1 H4.V alleles are knocked out and ectopic overexpression of a Ty1-tagged version of H4.V can be induced has previously been published21.The cell line in which both endogenous H3.V32 and ΔH4.V21 cells had been generated in a ‘single marker’ background34, we generated the ΔH3.VΔH4.V cells in a single marker background and compared the Hi-C profiles of the transgenic cell lines to those generated from single marker cells. Thus, all ‘wild-type’ Hi-C data shown in Figs. Because Δ36 and adapted to T. brucei: 2 × 108 cells  were collected and resuspended in 40 ml of 1× trypanosome dilution buffer . Cells were fixed in the presence of 1% formaldehyde for 20 min at room temperature by addition of 4 ml of formaldehyde solution . The reaction was stopped by addition of 3 ml of 2 M glycine and incubation for 5 min at room temperature and 15 min on ice. Cells were washed twice in 1× TDB and the cell pellet was snap-frozen in liquid nitrogen. Cells were resuspended in 1 ml of permeabilization buffer  supplemented with protease inhibitors  and digitonin  and incubated for 5 min at room temperature. Cells were washed twice in 1× NEBuffer3.1  and resuspended in 342 μl of 1× NEBuffer3.1. After addition of 38 μl of 1% SDS, and an incubation at 65 °C for 10 min, the SDS was quenched by addition of 43 μl of 10% Triton-X 100 (Sigma) and the incubation was continued at room temperature for 15 min. Another 35 μl of water, 13 μl of 10× NEBuffer3.1 and 100 units of MboI  were added and the chromatin was digested at 37 °C overnight while shaking. To inactivate MboI, the sample was incubated at 65 °C for 20 min. Restriction fragments were biotinylated by supplementing the reaction with 60 μl of fill-in mix , 0.25 mM dCTP, 0.25 mM dGTP, 0.25 mM dTTP (Fermentas), 40 U of DNA polymerase I, large (Klenow) fragment ) and incubation at 23 °C for 4 h. The end-repaired chromatin was transferred to 665 μl of ligation mix  and 5 μl of T4 DNA ligase  were added. The ligation was performed for 4 h at 16 °C with interval shake. Crosslinks were reversed by adding 50 μl of 10 mg/ml proteinase K (65 °C for 4 h) and another addition of 50 μl of 10mg/ml proteinase K, 80 μl of 5M NaCl and 70 μl of 10% SDS .In situ Hi-C was performed based on previously published protocolsThe DNA was precipitated with ethanol and resuspended in 257 μl of TLE . SDS was added to a final concentration of 0.1% and the sample was split among two tubes for sonication . The samples were recombined and the volume was adjusted to 300 μl with TLE. Fragments between 100 and 400 bp in size were selected using Agencourt AMPure XP beads (Beckman Coulter), according to the manufacturer’s instructions. The DNA fragments were eluted off the beads in 55 μl of TLE.For end-repair and biotin removal from unligated ends, 70 μl of end-repair mix was added (1× Ligation buffer (NEB), 357 μM dNTPs, 25U T4 PNK , 7.5U T4 DNA polymerase I , 2.5U DNA polymerase I, large (Klenow) fragment ) and incubated for 30 min at 20 °C and 20 min at 75 °C. To inactivate the enzymes, EDTA was added to a final concentration of 10 mM. To isolate biotin-labelled ligation junctions, 50 μl of 10 mg/ml Dynabeads MyOne Streptavidin C1  were washed with 400 μl of 1× Tween washing buffer , collected with a magnet, resuspended in 400 μl of 2× binding buffer  and added to the sample suspended in 330 μl TLE. Biotinylated DNA was bound to the beads by incubating the sample for 15 min at room temperature with slow rotation. Subsequently, the DNA-bound beads were captured with a magnet, washed twice with 400 μl of 1× binding buffer, washed once in 100 μl of 1× TLE T4 ligase buffer and resuspeded in 41 μl of TLE. For polyadenylation, 5 μl of 10× NEBuffer 2.1, 1 μl of 10 mM dATP and 3 μl of 5 U/μl of Klenow fragment (3′→ 5′ exo-)  and incubated for 30 min at 37 °C followed by deactivation for 20 min at 65 °C. Beads were reclaimed with a magnet, washed once with 400 μl 1× Quick ligation buffer  and resuspended in 46.5 μl of 1× Quick ligation buffer . 2.5 μl of DNA Quick ligase  and 0.5 μl of 50 μM annealed TruSeq adapters were added and incubated for 1 h at room temperature. Beads were separated on a magnet, resuspended in 400 μl of 1× TWB  and washed for 5 min at room temperature with rotation. Beads were washed on the magnet with 200 μl 1× binding buffer and 200 μl of 1× NEBuffer 2.1 and resuspended in 20 μl of 1× NEBuffer 2.1. The library was amplified in 8 separate reactions of 50 μl. Per reaction, 1.5 μl of 25 μM TruSeq PCR primer cocktail , mapped using bwa mem (https://arxiv.org/abs/1303.3997) of the bwa kit version 0.7.12-r1039 and those with a quality score q > 0 were retained. Reads were mapped to the T. brucei Lister 427 genome version 9 (Tb427v9). For each chromosome, this genome contains the core region of two homologous chromosomes only once, whereas both of the the respective heterozygous subtelomeric regions belonging to the two homologous chromosomes are included. During the mapping of Hi-C reads, contigs that displayed alternative variations of an assembled allele were removed  to keep these loci visible in the Hi-C matrices. The primary analysis of Hi-C reads was performed with HiC-Pro38 (version 2.10.0) to visually inspect reproducibility among replicates. Raw matrices were normalized for differences in ploidy , balanced by iterative correction using HiC-Pro (default settings) and converted into a homer compatible format39 using a custom Python script (see ‘Code availability’). To enable comparisons between different Hi-C experiments, each value in the balanced interaction matrix was divided by the respective column sum.Reads were trimmed at their ligation junction using the truncator of the HiCUP pipelineTo visualize the distance-dependent decay, interaction frequencies between Hi-C bin pairs were grouped on the basis of the linear distance between the pairs, and the distribution of the median interaction frequency across distances was plotted.t-test.To determine whether a region of interest interacted more or less than expected by chance, the median and mean interaction frequencies were calculated for bins overlapping with the regions of interest. In addition, a ‘background’ interaction frequency was determined by randomly selecting regions of identical size from the same matrix. Significance was determined using Welch’s H3.V, ΔH4.V and ΔH3.VΔH4.V cells. Significance was determined using Welch’s t-test. Co-localization analyses were performed using balanced interaction matrices (50-kb resolution). Bins with zero values were excluded from the analyses.To identify changes in DNA–DNA interaction frequencies after deletion of histone variants, the ratio of ‘feature median interaction frequencies’/‘background median interaction frequency’ was determined for 100 randomly selected background regions in wild-type, ΔTo visualize interactions between one genomic region (bait) and all other genomic sites, relevant bins were extracted from a 20-kb Hi-C matrix. An average interaction value for every genomic bin was calculated if the bait regions spanned more than one bin.https://seaborn.pydata.org). To generate differential heat maps, a pseudo-count of 0.000001 was added to each interaction value of the numerator and denominator matrix before division.Matrices were plotted based on the colour palettes provided by seaborn (8 cells of the T. brucei 427 17.13 P10 isolate40 using the Blood & Cell Culture DNA Midi Kit (Qiagen), according to the manufacturer’s instructions. In addition, the DNA was purified in a phenol chloroform extraction using Manual Phase Lock Gel 2-ml (heavy/light) tubes (5Prime) and suspended in 100 μl of TE buffer. SMRT library preparation and sequencing was performed at the Icahn School of Medicine at Mount Sinai.Genomic DNA was isolated and precipitated from 3 × 10Genomic DNA library preparation and sequencing was performed primarily using the manufacturer’s instructions for the P6-C4 sequencing enzyme and chemistries. In short, ~5 μg gDNA was quantified and diluted to 150 μl using elution buffer (Qiagen) at 33 ng/μl and then sheared to ~20 kb by centrifugation at 4,500 rpm for 50 s using a G-tube spin column (Covaris). The sheared DNA was then re-purified using Agencourt AMPure XP beads (Beckman Coulter) at 0.45×. Next, ~1.6 to 3.2 μg of DNA from each batch was taken into DNA damage and end repair. In brief, the DNA fragments were repaired by adding 21.1 μl of DNA damage repair solution  and 1× DNA damage repair mix) and incubation at 37 °C for 20 min. DNA ends were repaired by adding 1× end repair mix to the solution and incubation at 25 °C for 5 min, followed by an additional 0.45× Agencourt AMPure XP purification step. Next, 0.75 μM of blunt adaptor was added to the DNA and 1× template preparation buffer (0.05 mM ATP and 0.75 U/μl T4 DNA ligase) was added to a final volume of 47.5 μl. This solution was incubated at 25 °C overnight, followed by incubation at 65 °C for 10 min to inactivate the ligase. To remove un-ligated DNA fragments, exonuclease cocktail (1.81 U/μl Exo III 18 and 0.18 U/μl Exo VII) was added to the library followed by a 60 min incubation at 37 °C. Two additional 0.45× Agencourt Ampure XP purification steps were performed to remove <2,000-bp molecular weight DNA and organic contaminants.The size of the library was validated using an Agilent DNA 12000 chip. Before P6-C4-based sequencing, Blue Pippin size selection was applied to remove molecules <7,000 bp. This step was conducted using Sage Science Blue Pippin 0.75% agarose cassettes to select libraries in the range of 7,000–50,000 bp. Primers were annealed to the size-selected SMRTbell at a ratio of 20:1 with the full-length libraries by denaturation (80 °C for 2 min) and slow cooling (0.1 °C/s to 25 °C). The polymerase-template complex was bound to the P6 enzyme using a ratio of 10:1 polymerase to SMRTbell at 0.5 nM for 4 h at 30 °C and then held at 4 °C until ready for magnetic-bead loading. The magnetic-bead loading step was conducted at 4 °C for 60 min. The magnetic-bead-loaded, polymerase-bound SMRTbell libraries were placed onto the RSII machine at a sequencing concentration of 50 pM and configured for a 240-min continuous sequencing run.41 with default parameters. In brief, a sequence seeding dataset with the longest sequencing reads was pulled apart. The remaining reads were mapped onto them to obtain error-corrected reads through a consensus procedure. The error-corrected reads were assembled by traditional overlap layout consensus with a Celera Assembler42. Contig sequences were polished with Quiver and were further joined and extended using PBJelly 2 (PBSuite_15.2.20)43. Remaining sequence errors were corrected using iCORN244 with previously published and new gDNA Illumina data available under GSM2586510 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM2586510) and ERS1503958 (http://www.ebi.ac.uk/ena/data/view/ERS1503958), respectively.The 642,583 sequencing reads from wild-type gDNA (seven SMRT cells) were assembled into contigs following the RS_HGAP_Assembly.3 workflow from SMRT Analysis v2.3.0For genome scaffolding, all Hi-C reads were combined, mapped onto the error-corrected HGAPv3 contigs and a balanced 10-kb bin heat map matrix of DNA–DNA interactions was generated as previously described. For the scaffolding, only a subset of the matrix that contained contigs larger than 50 kb was considered  were broken at the site of the putative mis-assembly. Available pipelines for the scaffolding of contigs based on DNA–DNA interaction frequencies are not designed for genomes that contain large regions of heterozygosityPRJEB18945 (http://www.ebi.ac.uk/ena/data/view/PRJEB18945).For a comparison of different assembly strategies and an assessment of the genome quality, see T. brucei TREU 927 genome were transferred using Companion 47. In addition, VSG genes were annotated based on similarity (>90% coverage and >95% identity to a VSG gene) of the available VSGnome dataset from the Lister 427 strain10 with NCBI-BLAST+ (version 2.2.31+). Overlapping VSG-gene entries were merged and named after the entry with the best bit score. For the identification of putative novel MESs, the assignment of BESs to chromosome ends and the identification of centromeres, see Annotations from the 48 with minor alterations. For the immunoprecipitation of cell lines with Ty1-tagged proteins , 50 μl of Dynabeads protein G (ThermoFisher) was separated on a magnet, the supernatant was removed and the beads were resuspended in 200 μl of PBS-Tween (0.02%) containing 10 μg of BB2 antibody49, and then incubated with slow rotation at 4 °C overnight. Antibody-coupled protein G beads were separated on a magnetic rack and washed three times with PBS-Tween (0.02%). Five hundred microlitres of ChIP sample was added, and incubated at 4 °C overnight with slow rotation. For ChIP assessment of the distribution of histone variants, the DNA was fragmented using micrococcal nuclease; for ChIP assessment of the distribution of SCC1, the DNA was sheared using a Covaris S220 instrument before target protein binding. Sequencing reads were mapped using bwa mem and coverage plots were generated using COVERnant (v.0.3.2) (https://github.com/konrad/COVERnant)13.Assays were performed as previously described6 and 2 × 106 cells. To this end 3 × 107 cells were collected and washed in 30 ml of cold 1× TDB. The pellet was resuspended in 300 μl permeabilization buffer with protease inhibitors, 3 μl of 4 mM digitonin was added and incubated for 5 min at room temperature. The cells were pelleted, resuspended in 600 μl isotonic buffer with protease inhibitors and split in two samples, containing 1 × 107 and 2 × 107 cells, respectively. The transposition reaction was performed by adding 50 μl of transposition mix to the pellet (25 μl TD (2× reaction buffer from Nextera kit), 25 μl TDE1 (Tn5 transposase from Nextera kit), 22.5 μl nuclease-free water) and incubation for 30 min at 37 °C. For the gDNA control, 200 ng of gDNA was treated in the same manner. The DNA samples were purified using Qiagen MinElute PCR Purification Kit and eluted in 10 μl EB . The transposed DNA fragments were amplified using the NEBNext High-Fidelity 2× PCR Master Mix (M0541) supplied with 2.5 μl of 25 μM barcoded primers and amplification for 13 cycles. The libraries were purified using AMPure XP beads (Beckman Coulter) according to the manufacturer’s instructions. The library fragment sizes between 150 and 1,000 bp were purified from a 6% polyacrylamide gel. Paired-end 76-bp sequencing was carried out using the Illumina NextSeq 500 system with a high-output NextSeq 500/550 kit, according to the manufacturer’s instructions. Sequencing reads were mapped using bwa mem and coverage plots were generated using COVERnant (v.0.3.2) (https://github.com/konrad/COVERnant)13.To ensure reproducibility of the assays independent of cell-number variations, all assays were performed with 1 × 10T. brucei wild-type and ΔH3.VΔH4.V cells were sorted  using a FACSAria III . A forward-scatter area versus side-scatter area plot was used to gate and sort the cells. T. brucei cells were sorted in 48-well plates (Brand) filled with 2.6 μl of 1× lysis buffer (Takara) supplemented with 0.01 μl of RNase inhibitor . Immediately after sorting, cells were placed on ice for 5 min and stored at −80 °C.6 (scRNA-seq I) or a 1:20 × 106 (scRNA-seq II) dilution of ERCC Spike-In Control Mix 1 . Libraries were prepared using SMART-Seq v.4 Ultra Low Input RNA Kit (Takara) using a quarter of the reagent volumes recommended by the manufacturer. PCR amplification was performed using 26 cycles (scRNA-seq I) or 22 cycles (scRNA-seq II), according to the supplier’s recommendations. cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter) and recovered in 15 μl of elution buffer (Takara). Libraries were quantified using the Qubit 3 Fluorometer with dsDNA Hs Assay kit (Life Technologies) and the quality of the libraries was assessed using a 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent)  using a quarter of the recommended volumes, 10 min for tagmentation at 55 °C and 1 min extension time during PCR amplification. Libraries were pooled (96 libraries for NextSeq) and sequencing was performed in paired-end mode for 2 × 75 cycles using Illumina’s NextSeq 500. Details of the sequencing results are listed in Supplementary Table Lysates from 50 trypanosomes and single trypanosomes were supplemented with 0.2 μl of a 1:2 × 1051 (version 1.8) and MarkDuplicates tool (2.18.3-SNAPSHOT) from Picard (http://broadinstitute.github.io/picard/), and the read counts-to-features were done with bedtools52 (version 2.26.0). To assess the quality of the data, for each scRNA-seq experiment the read counts to the following groups were determined: reads mapping to ‘rRNA genes’, reads mapping to ‘protein-coding genes’ (CDS plus 89 bp for the 5′ UTR and 400 bp for the 3′ UTR)53, reads mapping to ‘other regions of the genome’ and ‘unmapped reads’. Reads that overlapped ‘rRNA genes’ and ‘protein-coding genes’ features at the same time were excluded from both groups and counted as reads mapping to ‘other regions of the genome’.The reads were mapped to the combination of the Tb427v9 genome with the ERCC spike-in sequences, using bwa mem, version 0.7.16. The mapped data were processed using samtoolsTo assess the library complexity of the scRNA-seq datasets, the number of genes with ≥ 10 read counts was determined for each cell. The counts per feature group, as well as the number of genes captured in each scRNA-seq experiment, are available in Supplementary Table Many VSG genes share a high degree of homology with each other. Therefore, to determine the expression levels for each of the 2,846 VSG genes annotated in Tb427v9, the number of uniquely mapping sequence reads obtained for each VSG gene was normalized to account for differences in uniqueness. The uniqueness of the VSG genes was determined by alignment of an in silico-generated dataset that matched the scRNA-seq datasets in read size and fragment-length distribution. For each cell, the transcript level of an individual VSG gene is shown as a percentage of the total VSG gene transcript level in that cell. Raw and normalized read counts are available in Supplementary Table T. brucei wild-type, ΔH3.V, ΔH4.V and ΔH3.VΔH4.V cells were grown to a density of ~106 cells/ml. Cell concentration was determined nine times for each replicate using a Coulter Cell Counter (Beckman Coulter) and 4.5 × 107 cells were collected from each culture. Cells were washed with 1× TDB, resuspended in 350 μl of buffer RA1  and 38 μl of 0.1 M DTT and 1 μl of a 1:10 dilution of ERCC Spike-In Control Mix  were added. Total RNA was purified from the lysate according to the NucleoSpin RNA kit protocol, and eluted in 30 μl of nuclease-free water. To deplete rRNA, 3.5 μg of total RNA was mixed with 2.6 μl of 5× hybridization buffer , 0.459 pmol of 131 50-bp anti-rRNA oligonucleotides  and 10 U of RNaseH  were added and the volume was adjusted to 16 μl with nuclease-free water. The mixture was incubated at 37 °C for 20 min. Residual oligonucleotides were subsequently digested by addition of 2 U of Turbo DNaseI and 5 μl of 10× Turbo DNase reaction buffer  in a total reaction volume of 50 μl and incubation at 37 °C for 20 min. The DNase was inactivated by addition of EDTA  and heating at 75 °C for 10 min. The RNA was purified using RNAeasy Minelute columns (Qiagen), according to the manufacturer’s instructions, and eluted in 14 μl of RNase-free water. Double-stranded cDNA was synthesized from 100 ng of rRNA-depleted RNA using the NEBNext Ultra RNA Library Prep Kit for Illumina. The double-stranded cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter) and eluted in 60 or 30 μl of 0.1× TE Buffer, respectively. Libraries were prepared and sequenced as previously described48.Triplicates of 54 (version 1.10), the RNA-seq reads were mapped against the T. brucei genome using bwa mem of the bwa kit version 0.7.16. Only reads with a quality score q > 0 were retained. Feature quantification was performed with bedtools multicov subcommand. Differential gene expression analysis was then conducted with DESeq2 (v.1.20.0)55. Features with an adjusted P value  below 0.1 were considered as differentially expressed. Supplementary Table 2 scale) and P value adjusted for each sample versus wild type.After adaptor clipping and quality trimming using cutadapt version .10, the 7 bloodstream-form trypanosomes grown to a density of up to 1 × 106 cells/ml were collected and the cell pellet was washed once with 1× TDB and fixed for 15 min in 1× TDB containing 4% formaldehyde. Cells were washed with 1× TDB once and resuspended in 50 μl of 1× TDB. Gene frames were placed onto microscopy slides to cover an aminopropyltriethoxysilane-coated coverslip. Cells were pipetted onto the framed coverslip and allowed to settle for 5 min by gravity. The sample was washed twice for 2 min with 90 μl of 1× TDB, incubated with quenching solution (1× TDB containing 1 mg/ml NaBH4) for 10 min, washed twice with 1× TDB, permeabilized with 70 μl of 1× TDB containing 0.1% NP-40 for 5 min and washed twice with 1× TDB. Next, cells were treated with 1× PBS containing 1 mg/ml RNaseA for 30 min, washed twice with 1× TDB and incubated with 50 μl of a 1:1 dilution of hybridization buffer with 2× SSC for 30 min at room temperature. The labelled probe  were suspended in HMI-11 containing 2% formaldehyde, incubated for 5 min at room temperature and washed with 1× TDB. α-Tubulin was stained using the mouse monoclonal antibody Tat158 (1:200) and a secondary Alexa Fluor 594-conjugated chicken anti-mouse IgG . VSG-2 was stained using CRD-depleted rabbit anti-VSG-240  and a secondary Alexa Fluor 488-conjugated donkey anti-rabbit IgG .Immunofluorescence was performed as previously described3 voxel size) were captured using identic exposure times for all conditions.For imaging, a wide-field fluorescence Leica DMI6000 microscope with a mercury metal halide lamp and a HCX PL APO CS 100×/1.47 OIL objective was used. Images were captured with a Leica DFC 360 FX camera. Stacks with 32 slices and 6.3232 μm in height . After segmenting individual nuclei in the DAPI channel, surfaces were rendered for the telomere FISH signal, while setting the quality filter > 1,000. All FISH signals that generated surfaces with a volume > 0.3 μm40. In brief, 1 × 106 cells were centrifuged in a chilled microcentrifuge at 1,500g for 4 min at 4 °C. Cells were resuspended in 100 μl of ice cold HMI-11 and a VSG-specific antibody (anti-VSG-2 or anti-VSG-13)40 was added. After 60 min of incubation at 4 °C with gentle shaking, cells were washed three times in 500 μl of ice cold HMI-11, resuspended in 100 μl of cold HMI-11 and incubated with an Alexa Fluor 488-conjugated secondary antibody for 20 min. The cells were washed twice with 500 μl of 1× TDB and finally resuspended in 400 μl of 1× TDB before analysis with a FACSort flow cytometer (Becton Dickinson Biosciences).VSG expression on the cell surface was quantified according to a previously published protocol6 cells were collected  and washed once with ice-cold 1× TDB. The cells were resuspended in 1 ml ice-cold PBS and 2 mM EDTA and fixed by adding 2.5 ml ice-cold methanol. After a 1-h incubation, cells were washed with 1 ml PBS and EDTA at room temperature and resuspend in 1 ml PBS and EDTA. One microlitre RNaseA (10 μg/μl) and 10 μl propidium iodide (1 μg/μl) were added. The stained cells were analysed with a FACSCalibur (Becton Dickinson) after a 30-min incubation at 37 °C.To determine the cell-cycle profiles, 5 × 10H3.VΔH4.V gDNA was prepared as described for wild-type gDNA and sequenced using the PacBio Sequel system by diffusion at 5 pM, with v2.1 chemistry and a 10-h movie. Reads > 10 Kb were extracted, split into ~2,500-bp chunks and mapped independently to the genome. Reads that contained chunks that mapped to BES1 and BES15 were kept and mapped again to BES1 and BES15. Based on the observed mapping pattern, a BES1–BES15 hybrid was constructed.A library from ΔWorkflows and custom-made Unix Shell, Python and R scripts have been deposited at Zenodo (10.5281/zenodo.823671). Documentation to reproduce the data analysis is provided.Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, statements of data availability and associated accession codes are available at 10.1038/s41586-018-0619-8.Supplementary InformationThis file contains details on genome assembly.Reporting SummarySupplementary Table 1RNA-seq analysis.Supplementary Table 2Single-cell RNA-seq analysis.Supplementary Table 3Genome quality assessment.Supplementary Table 4Oligo lists.