Tg) and thermal decomposition temperature was prepared by simple thermal crosslinking of PMMA-containing random copolymers bearing aryl trifluorovinyl ether (TFVE) moieties. A methacrylate monomer consisting of aryl TFVE moiety, 4-oxy)phenyl methacrylate (TFVOPMA), was first synthesized followed by radical copolymerization with methyl methacrylate (MMA) initiated by AIBN, providing the random copolymer containing aryl TFVE moieties, polyoxy)phenyl methacrylate)-co-poly(methyl methacrylate) (PTFVOPMA-co-PMMA). Finally, crosslinked PMMA polymer with perfluorocyclobutyl (PFCB) aryl ether moieties as crosslinking units was obtained by [2π + 2π] cycloaddition reaction of aryl TFVE moieties in PTFVOPMA-co-PMMA copolymer. Thermal properties of both PTFVOPMA-co-PMMA and crosslinked PTFVOPMA-co-PMMA were examined by TGA and DSC. Compared to pure PMMA, Tg of PTFVOPMA-co-PMMA increased by 15.1 °C and no Tg was found in the DCS test of the crosslinked PTFVOPMA-co-PMMA. Thermal decomposition temperature  of crosslinked PMMA was 47 °C higher than that of pure PMMA. Furthermore, the water absorption of crosslinked PMMA film greatly reduced in comparison with that of pure PMMA.Crosslinked poly(methyl methacrylate) (PMMA) with high glass transition temperature ( PMMA-based copolymer containing aryl TFVE moieties prepared by copolymerization of MMA and TFVOPMA monomer containing aryl TFVE group and thermal cross-linked by [2π + 2π] cycloaddition reaction of aryl TFVE moieties. Wilkie et al. prepared MMA–DVB crosslinked polymers using divinylbenzene (DVB) as crosslinking agents and the degradation temperature  of resulting polymers increased from 162 °C to 294 °C.12 Kuo et al. suggested an approach to raise the Tg of PMMA through copolymerization with methacrylamide (MAAM) since hydrogen-bonding interactions exist between these two monomer segments.16–19Poly(methyl methacrylate) (PMMA) is a kind of transparent polymeric material possessing diverse excellent properties such as superior light transmittance, light weight, chemical stability, weathering corrosion resistance, electrical insulation and good processability, which make PMMA widely applied in many fields including aerospace, building construction and optical instruments.20–22 To avoid low processability of perfluoropolymers, partially-fluorinated polymers like poly(vinylidene fluoride) (PVDF),4,5 alternating ethylene-tetrafluoroethylene (ETFE)23 and alternating ethylene-chlorotrifluoroethylene (ECTFE) copolymers,24,25 have been prepared and exhibited improved processability. Among partially-fluorinated polymers, perfluorocyclobutyl (PFCB) aryl ether polymers26–31 are an intriguing class of fluoropolymers, which not only retain the general outstanding properties of fluoropolymers originating from the low polarity, strong electronegativity and small van der Waals radius of fluorine atom and strong C–F bond, but also possess many other advantages such as improved processability and optical transparency, structural versatility and tunable properties. These PFCB aryl ether-based polymers have been studied on the potential application in photonics, polymer light-emitting diodes, proton exchange membrane in fuel cell and atomic oxide resistance materials on spacecraft etc.32–40Fluoropolymers have attracted much curiosity in material science due to their unique properties including thermal stability, chemical resistance, flame retardancy, superior electrical insulating ability, low dielectric constant and refractive index, and unique surface property.et al. prepared a series of PFCB-containing copolymers with high heat resistance.41–43 The resulting polymethacrylate bearing sulfonyl functionality exhibits excellent thermal stability (Td > 300 °C) and polyimide (PFCBBPPI) containing PFCB biphenyl ether moieties shows excellent thermal stability and high transparency. PFCB aryl ether polymers are most commonly prepared through a thermal [2π + 2π] cycloaddition of aryl trifluorovinyl ethers (TFVE) at temperatures of between 150 °C and 200 °C without additional agent and no condensation byproducts are produced. Thus, TFVE group may act as a crosslinkable group and the crosslinked region, perfluorocyclobutyl (PFCB) moieties, may give rise to better chemical resistance. Lee et al. synthesized a novel fluorinated aromatic polyether monomer containing TFVE group and the resulting crosslinked polymers after thermal crosslinking showed higher Tg (239–271 °C).44Inspired by the fact that PFCB aryl ether-based polymers possess high heat resistance and optical transparency, Prof. Huang of Shanghai Institute of Organic Chemistry Tg and high Tdvia copolymerization of MMA with a methacrylate monomer containing TFVE moiety, 4-oxy)phenyl methacrylate (TFVOPMA), followed by thermal crosslinking process as shown in All aforementioned discussions intrigued us to synthesize methacrylate monomer containing TFVE group, which can be copolymerized with MMA to provide polymethacrylate copolymers containing TFVE groups so that the subsequent thermal cycloaddition of TFVE groups can produce crosslinked polymethacrylate copolymers with PFCB aryl ether moieties as the crosslinked region. The resulting crosslinked polymethacrylate copolymers may have higher thermal stability and good optical transparency because of the below points: (1) PFCB aryl ether-based polymers exhibit higher heat resistance and good transparency, (2) PFCB aryl ether moieties with large volume may decrease the activity of PMMA main chain, which might increase the heat resistance of resultant polymers, (3) three-dimensional network structure may limit the mobility and rotation ability of polymeric chain, which benefits heat resistance of resultant polymers. Herein, we report the preparation of transparent crosslinked poly(methyl methacrylate) (PMMA) copolymers with high 2 and distilled under reduced pressure prior to use. Triethylamine  was dried over KOH and distilled over CaH2 under N2 prior to use. Granular zinc was activated by washing in 0.1 N HCl followed by drying at 140 °C in vacuo for 10 h. 2,2′-Azobis(isobutyronitrile)  was recrystallized from anhydrous ethanol. Dichloromethane (DCM) and acetonitrile was dried over CaH2 and distilled under N2 prior to use. 2-Butanone was refluxed with KMnO4 and distilled under CaCl2 and KMnO4 prior to use. Methyl methacrylate  was washed with 5% aqueous NaOH solution to remove the inhibitor, then washed with water, dried over CaCl2 and distilled twice in vacuo from CaH2 prior to use. 1,2-Dibromotetrafluoroethane was prepared by condensing equimolar amounts of Br2 and tetrafluoroethylene at −195 °C followed by warming up to 22 °C according to a previous report.1 4-methoxylphenol , caesium carbonate , boron tribromide  and methacryloyl chloride  were used as received unless otherwise specified.Dimethylsulfoxide  was dried over CaH1H, 13C and 19F NMR spectra of intermediates were recorded on a JEOL resonance ECZ 400S spectrometer (400 MHz) in CDCl3. Tetramethylsilane (TMS) and CDCl3 were used as internal standards for 1H and 13C NMR, respectively; CF3CO2H was used as an external standard for 19F NMR. FT-IR spectra were recorded on a Nicolet AVATAR-360 FT-IR spectrophotometer with a resolution of 4 cm−1. Number-average molecular weights (Mn) and molecular weight distributions (Mw/Mn) were obtained on a conventional gel permeation chromatography (GPC) system equipped with a Waters 515 Isocratic HPLC pump, a Waters 2414 refractive index detector, and a set of Waters Styragel columns (HR3 (500–30 000), HR4 (5000–600 000) and HR5 (50 000–4 000 000), 7.8 × 300 mm, particle size: 5 μm). GPC measurement was carried out at 35 °C using tetrahydrofuran (THF) as eluent with a flow rate of 1.0 mL min−1. The system was calibrated with linear polystyrene standards. Differential scanning calorimetry (DSC) was performed on a TA Q200 DSC instrument in N2 with a heating rate of 10 °C min−1. Thermogravimetry analysis (TGA) was conducted on a TA Discovery TGA 55 thermal analysis system in N2 with a heating rate of 10 °C min−1. UV/vis spectra were acquired on a Hitachi U-2910 spectrophotometer.via four steps starting from 4-methoxylphenol  was added via a gastight syringe and the mixture was stirred for 30 min. 1,2-Dibromotetrafluoroethane  was then dropped slowly, keeping the temperature below 35 °C. The resulting mixture was heated at 50 °C for 5 h. Finally, 500 mL of deionized water was added and the organic layer was separated and extracted by dichloromethane, washed by water and brine and then dried over magnesium sulfate (MgSO4). 1-Methoxy-4-benzene  was obtained by silica column chromatography using hexane as eluent. 1H NMR : δ (ppm): 7.23 , 7.03 , 3.82 . 19F NMR : δ (ppm): −68.2 , −86.6 . 13C NMR : δ (ppm): 55.7, 114.6, 122.8, 142.0, 155.3. EI-MS (M+): 123 (100), 287 (1), 289 (1).4-oxy)phenyl methacrylate (TFVOPMA) was synthesized ylphenol . 1-Metho3  dropwise within 30 min. The mixture was slowly warmed up to room temperature and stirred at room temperature for another 12 h. The resulting mixture was filtrated and the residual salt was removed by washing with dichloromethane and filtrating three times. The solvents were removed by rotary evaporation, and the residue was subjected to column chromatography (eluent: hexane) to afford 4-phenol as a colorless liquid . 1H NMR : δ (ppm): 7.10 , 6.86 , 5.73 . 19F NMR : δ (ppm): −68.2 , −86.27 . 13C NMR : δ (ppm): 116.3, 122.9, 142.0, 154.4. EI-MS (M+): 109 (100), 356 (2), 358 (2).In a pre-dried flask, 1-methoxy-4-benzene  was dissolved in 300 mL of anhydrous dichloromethane. The solution was cooled to 0 °C followed by adding dichloromethane solution of BBr4. The organic layer was filtrated and evaporated under reduced pressure, and then the residue was purified by flash column chromatography (eluent: hexane) on silica gel to give 3.79 g (82%) of 4-phenyl methacrylate as a colorless oil. 1H NMR : δ (ppm): 7.12 , 7.06 , 6.24  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>CHH), 5.66 , 1.94 . 19F NMR : δ (ppm): −70.02 , −88.01 . 13C NMR : δ (ppm): 18.4, 122.7, 123.1, 127.7, 135.6, 145.9, 149.3, 165.6.In a pre-dried flask, 4-phenol  and triethylamine  were dissolved in 300 mL of 2-butanone. The solution was cooled to 0 °C followed by adding the solution of methacryl chloride  in 10 mL of 2-butanone dropwise. The mixture was warmed up to room temperature and stirred for another 1 h. The salt was removed by filtration and the filtrate was washed with water and dried over MgSO2 three times. Next, dry acetonitrile (40 mL) was added and the oil temperature was lifted to 90 °C. 4-phenyl methacrylate  was dropped slowly into the above mixture, keeping the mixture boiling slightly. The mixture was refluxed for 10 h and after cooled to room temperature, the mixture was filtered. The salt was washed by dichloromethane to extract the product. The organic phase was collected and dried over MgSO4. After CH2Cl2 was evaporated, the crude product was purified by flash column chromatography (eluent: hexane) to afford 4-oxy)phenyl methacrylate (TFVOPMA) as a colorless oil . 1H NMR : δ (ppm): 7.23 , 7.17 , 6.31 , 5.75 , 2.03 . 13C NMR : δ (ppm): 18.1, 116.7, 123.2, 127.2, 135.6, 147.7, 165.6. 19F NMR : δ (ppm): −120.1 , −126.9 , −134.49 . FT-IR (KBr): ν (cm−1): 2962, 2936, 2856, 1832, 1740, 1640, 1501, 1318, 1276, 1181, 1140, 947, 876, 812, 752.Newly activated zinc  was added to a dried three-neck round-bottom flask followed by evacuating and backfilling with Nvia a gastight syringe. The solution was degassed by three cycles of freezing–pumping–thawing followed by immersing the flask into an oil bath preset at 60 °C to start the polymerization. The polymerization was terminated by putting the flask into liquid nitrogen after 3 h. After repeated purification by dissolving in 2-butanone and precipitating in ethanol three times, 1.39 g (78%) of white powder, polyoxy)phenyl methacrylate)-co-poly(methyl methacrylate) (PTFVOPMA-co-PMMA), was obtained after drying in vacuo at 30 °C. GPC: Mn = 50 900 g mol−1, Mw/Mn = 1.84. 1H NMR : δ (ppm): 0.84, 1.02, 1.18 , 1.85–2.04 , 3.58 , 7.11 . 19F NMR : δ (ppm): −119.2 , −126.1 , −134.0 . FT-IR (KBr): ν (cm−1): 2998, 2947, 2845, 1834, 1731, 1499, 1299, 1138, 984, 838, 812, 751.AIBN  was firstly added to a 25 mL Schlenk flask (flame-dried under vacuum prior to use) sealed with a rubber septum for degassing. Next, TFVOPMA , MMA  and 2-butanone (3.4 mL) were introduced co-PMMA copolymer was dissolved in ethyl acetate and the solution was then spin-cast onto the freshly cleaned glass substrate followed by heat treatment at 30 °C for 30 min, 40 °C for 30 min and 65 °C for 1 h. The obtained thin film was placed into a beaker and then the beaker was placed into a tube furnace, followed by evacuating and backfilling with N2 three times. The temperature of tube furnace was raised to 100 °C with a rate of 5 °C min−1. After keeping at 100 °C for 30 min, the temperature was raised to 150 °C with a rate of 5 °C min−1. After keeping at 150 °C for 30 min, the temperature was raised to 180 °C with a rate of 5 °C min−1. After reacting at 180 °C for 10 h, the furnace was cooled and crosslinked PTFVOPMA-co-PMMA copolymer film was obtained. FT-IR (KBr): ν (cm−1): 2998, 2950, 1732, 1501, 1488, 1452, 1276, 1199, 1150, 969, 842, 807, 752.PTFVOPMA-co-PMMA) were dried in vacuo at 55 °C for 24 h and a constant weight (±0.0001 g) can be obtained for the film (about 0.5 g). The films were immersed in ultra-pure water at 25 °C. At certain interval, the films were then taken out of water and the water absorbed on the surface was wiped off by a cleaning cloth. The weight of the films was weighed immediately.The water uptake experiments were carried out as follows: sample films phenol, which was treated with methacryloyl chloride to obtain 4-phenyl methacrylate. Finally, –OCF2CF2Br moiety of the methacrylate was converted to –OCFCF2 functionality by Zn-mediated elimination to give the target methacrylate monomer, 4-oxy)phenyl methacrylate (TFVOPMA).TFVOPMA, a methacrylate monomer containing aryl TFVE moiety, was synthesized 1H NMR and 19F NMR. The characteristic peaks of double bond are found to be located at 5.75 (‘b’) and 6.31 (‘c’) ppm in 1H NMR spectrum of TFVOPMA  (PTFVOPMA-co-PMMA).Compared to living/controlled polymerization, conventional free radical polymerization allows achieving higher molecular weights and the related industrial technology is mature. Therefore, free radical copolymerization of MMA and TFVOPMA was conducted in 2-butanone at 60 °C using AIBN as initiator to provide the corresponding random copolymer, polyoxy)phenyl methacrylate)-1H NMR and 19F NMR. GPC retention curve of PTFVOPMA-co-PMMA shows a unimodal elution peak with a relatively broad molecular weight distribution of 1.84 and the molecular weight of PTFVOPMA-co-PMMA is about 50 900 g mol−1 , 1499 , 812 and 838 (Ar–H deformation) are visible and the stretching vibration of –CH3 (2998 cm−1) and –CH2 (2947 and 2845 cm−1) become stronger, which also illustrate the preparation of PTFVOPMA-co-PMMA copolymer. 1H NMR spectrum of PTFVOPMA-co-PMMA copolymer, which also showed the disappearance of proton resonance signal of double bond. The peaks of polymethacrylate backbone appeared at 1.85–2.04 ppm (‘b’) and 0.84, 1.02, 1.18 ppm (‘a’), corresponding to the protons of methylene and methyl in –CH2CCH3 moiety. The peak at 3.58 ppm (‘c’) is attributed to 3 protons of methoxy group, while the peaks at 7.11 ppm (‘d’) belongs to the protons of phenyl. Therefore, the composition of this copolymer (NTFVOPMA/NMMA) was about 1 : 1.93, which was calculated according to the equation  can form perfluorocyclobutane units via [2π + 2π] cycloaddition reaction at high temperature, thus we firstly monitored this cross-linking reaction by DSC. As we can see from Tg of PTFVOPMA-co-PMMA was about 137.6 °C in round 1, higher than that of PMMA (122.5 °C). We supposed that the introduction of rigid benzene ring with large volume makes rotational hindrance of polymeric chain difficult, which leads to higher Tg of PTFVOPMA-co-PMMA. Interestingly, Tg of PTFVOPMA-co-PMMA increased with the scan times and the values of Tg reached 169.7 °C in the sixth scan. The phenomenon demonstrated that [2π + 2π] cycloaddition reaction of trifluorovinyl ether groups in PTFVOPMA-co-PMMA copolymer occured at higher temperature during the DSC measurement and the formed PFCB cross-linking unit further limited the mobility of polymer chains, endowing the polymer with higher Tg.Trifluorovinyl ether groups  for PMMA, PTFVOPMA-co-PMMA and crosslinked PTFVOPMA-co-PMMA are 254 °C, 250 °C and 301 °C, respectively; while the 10% weight loss temperatures  are 269 °C, 276 °C and 326 °C, respectively. PTFVOPMA-co-PMMA containing TFVE groups shows lower thermal degradation temperature at first stage before Td,6% (257.6 °C) as compared to that of PMMA, which may be attributed to the polarity and softness of TFVE group. However, PTFVOPMA-co-PMMA containing TFVE groups shows higher Td at late stage , which may be attributed to the formation of enough PFCB rings at higher temperature. As we can see, crosslinked PTFVOPMA-co-PMMA with PFCB aryl ether moieties as crosslinked units shows higher thermal stability compared to PMMA and PTFVOPMA-co-PMMA. This can be explained that the formation of PFCB ring after thermal treatment decreases the activities of PMMA main chain because of its large volume, and three-dimensional network structure limits the mobility and rotation ability of polymer chain, which may improve the thermal stability of the resultant PMMA-based copolymer.As it can be seen from co-PMMA and cross-linked PTFVOPMA-co-PMMA films were investigated by UV/vis spectroscopy. As shown in co-PMMA film are 74% at 500 nm and 80% at 600 nm, and the transmittance of the crosslinked film decreased to 62.5% at 600 nm.The light transmittance of PTFVOPMA-co-PMMA films after immersing into pure water for a certain time (co-PMMA film greatly decreased in comparison with pure PMMA (0.03% vs. 1.73% after 8 h). Furthermore, the water absorption of crosslinked PTFVOPMA-co-PMMA film retained below 0.06%, while that of PMMA increased with the immersing time. The decreasing water absorption could be due to the highly hydrophobic nature of aryl PFCB moieties.Among the unique properties of fluorinated polymers, low water absorption is a typical one. The moisture resistance was evaluated by measuring the weight change of PMMA and crosslinked PTFVOPMA-ain time . It is nco-PMMA, which was treated at 180 °C to give crosslinked network PTFVOPMA-co-PMMA copolymer via the formation of PFCB moieties. The glass transition temperature of crosslinked network PTFVOPMA-co-PMMA copolymer is high and they are thermally stable with less water absorption.In summary, a methacrylate monomer containing aryl TFVE group, TFVOPMA, was synthesized and copolymerized with MMA to provide the copolymer containing aryl TFVE groups, PTFVOPMA-There are no conflicts to declare.


V7.1/KCNE1, which is the most frequently mutated ion channel in LQTS.Genotype-positive patients who suffer from the cardiac channelopathy Long QT Syndrome (LQTS) may display a spectrum of clinical phenotypes, with often unknown causes. Therefore, there is a need to identify factors influencing disease severity to move towards an individualized clinical management of LQTS. One possible factor influencing the disease phenotype is the endocannabinoid system, which has emerged as a modulator of cardiovascular function. In this study, we aim to elucidate whether endocannabinoids target the cardiac voltage-gated potassium channel Kex-vivo guinea pig hearts.We used two-electrode voltage clamp, molecular dynamics simulations and the E4031 drug-induced LQT2 model of V7.1/KCNE1. Using the endocannabinoid ARA-S as a prototype, we show that the effect is not dependent on the KCNE1 subunit or the phosphorylation state of the channel. In guinea pig hearts, ARA-S was found to reverse the E4031-prolonged action potential duration and QT interval.We found a set of endocannabinoids that facilitate channel activation, seen as a shifted voltage-dependence of channel opening and increased overall current amplitude and conductance. We propose that negatively charged endocannabinoids interact with known lipid binding sites at positively charged amino acids on the channel, providing structural insights into why only specific endocannabinoids modulate KV7.1/KCNE1 channel modulators with putative protective effects in LQTS contexts.We consider the endocannabinoids as an interesting class of hK10.13039/100000190ERC (No. 850622), 10.13039/501100000024Canadian Institutes of Health Research, 10.13039/501100001804Canada Research Chairs and 10.13039/100013020Compute Canada, Swedish National Infrastructure for Computing. V7.1/KCNE1 channel is the most frequently mutated channel. However, there is individual variability in the disease severity of LQTS, which may be caused by endogenous factors that modulate ion channels.The heart rhythm is maintained by the electrical activity of several ion channels and transporters. Mutations in different ion channels have been linked to the arrhythmia Long QT Syndrome (LQTS), in which the KV7.1/KCNE1 channel and provide mechanistic insights into endocannabinoid binding sites and mechanisms of action. Moreover, we find that endocannabinoids restore a physiological QT interval in a guinea pig model of LQTS.We find that endocannabinoids are potent activators of the KCombined, these findings show that endocannabinoids may act as disease modifiers in LQTS.,,Congenital Long QT Syndrome (LQTS) is an inherited cardiac channelopathy characterised by a delayed ventricular repolarization.,,202 to synthesize ARA-S.,,V4.3 and KV1.5,,V and L-type CaV channels in ventricular myocytes,,V7.2/7.3 channel,,V channel.The endocannabinoid system has emerged as a putative modulator of cardiovascular function. This system includes the two best known lipid-based endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide V7.1/KCNE1, which is the most commonly mutated channel in LQTS, remains unstudied. KV7.1/KCNE1 generates the slowly activating component of the delayed rectifier K+ current (IKs) in cardiomyocytes, which contributes to cardiomyocyte repolarization and is essential for the physiological shortening of the APD and QT interval triggered by sympathetic stimulation of the heart.V7.1 and up to four auxiliary KCNE1 subunits.V7.1 has the general structure of voltage-gated K+ channels formed through tetramerization of channel subunits. Each subunit has 6 transmembrane segments (S1 to S6), of which S1-S4 form the voltage-sensing domains (VSD) and segments S5-S6 form a common pore domain (PD).+ currents.,V7.1/KCNE1 for interacting with different types of lipids, including the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2),V7.1/KCNE1 may be a non-canonical cardiac target of endocannabinoids.However, whether endocannabinoids also target the cardiac voltage-gated potassium channel KXenopus oocytes expressing the human KV7.1/KCNE1 (hKV7.1/KCNE1) channel, we found that negatively charged endocannabinoids, like ARA-S, target hKV7.1/KCNE1 and facilitate channel activation, seen as a shifted voltage-dependence of channel opening and increased overall current amplitude and conductance. Computer simulations together with experiments suggest that endocannabinoids interact with known PUFA binding sites at positively charged amino acids on the channel and provide mechanistic insights into why only negatively charged endocannabinoids are effective. The effect does not depend on the KCNE1 subunit or the phosphorylation state of the channel. In guinea pig hearts, ARA-S reverses drug-induced prolongation of the APD and QT interval. Hence, we consider the endocannabinoids as an interesting class of hKV7.1/KCNE1 channel modulators with putative protective effects in LQTS contexts.Using the two-electrode voltage clamp technique on Detailed materials and methods are described in the Xenopus laevis experiments were approved by the Linköping Animal Care and Use Committee (Permit #1941) and conform to national and international guidelines. Guinea pig experiments were performed at the Department of Biomedical Sciences, University of Copenhagen, Denmark and done according to the Danish guidelines for animal experiments under license 2017-15-0201-01296.Xenopus laevis oocytes were isolated and maintained as previously described.V7.1, KCNE1 and/or Ci-VSP as indicated in each results section. Two-electrode voltage clamp experiments were performed using a Dagan CA-1B amplifier  or an AxoClamp 900 A amplifier . All endocannabinoids were bought from Cayman Chemicals , except ACEA which was bought from Tocris Bioscience . DOC-S, LIN-S, ARA-D-S were synthesized in house as described previously.MIN is the minimum conductance, GMAX the maximum conductance, V50 the midpoint , and s the slope of the curve.To quantify the voltage dependence of channel opening, tail currents were measured shortly after stepping to the tail voltage and plotted against the preceding activation voltage. A Boltzmann function was fitted to the data to generate the conductance versus voltage G(V) curve:MAX is the maximal shift in V50, the maximal change in current amplitude or the maximal change in GMAX, EC50 the concentration needed to cause 50% of the maximal effect, and H the Hill coefficient (set to 1).To plot the concentration dependence of the compound-induced effect as a function of the compound concentration, the following concentration–response curve was fitted to the data:V7.1. As a starting structure, we used the Xenopus laevis KV7.1 structure, solved by cryo-EM at 3.7 Å (PDB: 5VMS).V7.1 numbering is used. Molecular dynamics simulations were performed using the transmembrane domain (residues 104-358) of the Cryo-EM structure of human KV7.1, solved at 3.1 Å.We used the Site-Identification by Ligand Competitive Saturation (SILCS) softwareThe Langendorff-perfused heart experiments were performed as described in Liin et al.,t test , Student's t test to compare between two groups or one-way ANOVA followed by Tukey's multiple comparisons test to compare multiple groups. For the isolated heart experiments, two-way ANOVA with Dunnett's multiple comparisons test was used. P < 0.05 was considered statistically significant. All statistical analyses were carried out in GraphPad Prism 9.Average values are expressed as mean ± SEM. Statistics were calculated using one-sample The funding sources had no involvement in the study design, the collection, analysis, and interpretation of data, and in the writing of the manuscript.V7.1/KCNE1 channel, we tested the effect of 10 μM of the endocannabinoids 2-AG, AEA, NADA, NAGABA, ARA-S, LIN-S and DOC-S . For GMAX, only one cell out of 6 responded with a noticeable GMAX increase, rendering a non-significant GMAX change of +65 ± 47%   a, modera 23 ± 4% , Table 196 ± 26% , Table 1.1/KCNE1 f and g, OC-S did f and g. trations . TherefoV7.1/KCNE1, seen as a shifted V50 and increased current amplitude, through direct binding to the KV7.1 channel.,,V7.1V7.1: One site (referred to as site 1) next to the S4 gating charges (R228) in the VSD, and the other (referred to as site 2) next to K326 in S6 in the PD.,V7.1 and a library of four ligands, i.e. arachidonic acid (ARA), LIN, ARA-S and LIN-S , compared to the GMAX increase in hKV7.1/KCNE1 WT  , compared to a V50 shift of −33.3 ± 5.6 mV  in hKV7.1 WT  d. Moreovby LIN-S e but maiX effect . 10 μM oKV7.1 WT e.V7.1_R293A and hKV7.1_D301A mutations. We co-expressed each mutant with KCNE1, because testing KV7.1_R293A alone produced negligible currents and KV7.1_D301A alone was previously reported to not generate currents.V7.1_K326Q/KCNE1 mutation, the GMAX effect induced by LIN-S was reduced in the hKV7.1_R293A/KCNE1 mutant compared to the GMAX effect in WT (t test). For the hKV7.1_D301A/KCNE1 mutant, the GMAX effect induced by LIN-S was completely abolished    f. On the 3.8 mV) . Hence, 50 and current amplitude of hKV7.1/KCNE1  , compared to a V50 shift of −39.1 ± 3.0 mV  in hKV7.1 WT  and increased the current amplitude at 0 mV    or alanine , or for uncharged head groups, such as serotonin  or serinol . Please refer to  t test) , Table 1ctively) , Table 150 = −29 ± 2.3 mV; ΔIamp = +587 ± 119%, V7.1/KCNE1. Moreover, the importance of the negative charge provides a mechanistic understanding why 2-AG, AEA, and NADA do not activate hKV7.1/KCNE1, as none of these compounds are negatively charged.To test if the ARA-S effect is also sensitive to the chirality of the head group, we used the unnatural D enantiomer of ARA-S  to the effect on hKV7.1/KCNE1. As mentioned above, 10 μM of ARA-S shifted V50 of hKV7.1 by −28 ± 2 mV and increased GMAX by 94 ± 25%  and hKV7.1_E160R_R237E (AO state) using a pulse protocol similar to previous studies.V7.1_E160R_R231E (IO state) and hKV7.1_E160R_R237E (AO state)  f. We not2, a phospholipid present in the inner leaflet of the cell membrane, is necessary for hKV7.1/KCNE1 function by mediating electromechanical VSD to PD coupling.,2 properties can act as a substitute for PIP2 and thereby compensate for PIP2 depletion.2, such as the lipid tail and negatively charged head group, and showed possible interactions in the inner leaflet in the simulations, we tested whether ARA-S can compensate for PIP2 depletion. To deplete PIP2 from the membrane, we followed previous protocols by co-expressing the voltage-dependent phosphatase Ci-VSP with hKV7.1/KCNE1 and activated the Ci-VSP with a consecutive depolarizing test pulse of +40 mV for 5 s followed by a tail pulse at −40 mV every 30 s.V7.1/KCNE1 generated clear K+ currents in response to the first depolarizing pulse (black line in left panel). As the phosphatase was activated and PIP2 depleted from the membrane, the initial hKV7.1/KCNE1 K+ current was gradually reduced (grey lines in left panel) with an exponential time-course of current run-down (time-course summary middle panel), meaning that fewer channels are able to open upon PIP2 depletion.PIP2 and prevent current run-down, oocytes were preincubated either in 10 μM extracellular . However, in oocytes with incomplete PIP2 depletion, ARA-S was able to partially recover the initial current amplitude . Combined, this suggests that ARA-S cannot substitute for PIP2 by binding to the PIP2 site to prevent current run-down. However, ARA-S can increase the current of the fraction of channels in which PIP2 is still present. Therefore, to some extent, it can functionally compensate for the reduced current caused by incomplete PIP2 depletion.To determine if ARA-S can substitute for PIPcellular b or intrcellular c ARA-S .,V7.1_S27D_S92D double mutant  of the left and right ventricle by 15.7 ± 3.7 ms and 15.8 ± 2.2 ms, respectively  in hKV7.1 to interact with the outermost S4 arginines R228 and R231 in site 1 (to facilitate outward S4 movement) and the S6 lysine K326 in site 2 (to improve K+ conductance).50 of related neuronal hKV7 channels.,V7.1 in the presence of LIN and LIN-S revealed important differences between how compounds with carboxylic and serine head groups interact with KV7.1. Overall, higher density for LIN-S was retrieved near the previously identified sites 1 and 2, interactions which were experimentally confirmed by the impaired effect of LIN-S on GMAX when mutating K326Q in site 2 and V50 when mutating R228Q in site 1. The analysis of hydrogen bond interactions revealed how the larger serine head group can establish hydrogen bonds with more residues compared to the carboxylic head group alone: At site 1, while LIN interacts with the previously reported R228 more than LIN-S, the larger serine head group interacts with residues deeper at the interface between the S4 segment of one monomer and the S5 segment of the neighbouring monomer . Similarly, at site 2, the simulations retrieve the interactions with K326 primarily for LIN, while LIN-S can interact with a larger number of residues closer to the pore domain and in the surrounding extracellular loops, particularly with D301 and R293. Thus, while sites 1 and 2 remain the primary regions of interactions of KV7.1 with PUFAs and endocannabinoids, distinct residues can control the effect of different compounds on modulating channel activity, which could be a possible explanation for why the hKV7.1_K326Q/KCNE1 mutation did not alter the GMAX effect of ARA-S. The improved ability of serine compounds to interact with KV7.1 in simulations compared to fatty acids, despite the same negative charge in the headgroup, suggests that the larger experimental effects induced by serine compounds are not only determined by different pKa values of the head groups (about 1 pH unit lower for a serine head group compared to a carboxylic head group). It is important to note that our simulations were done on KV7.1 alone, without the KCNE1 subunit. This is because the structure of the KV7.1/KCNE1 complex has not yet been determined. The presence of KCNE1 in the simulation systems may affect endocannabinoid distribution at the KV7.1 channel. However, because ARA-S and LIN-S show a generally similar experimental effect on KV7.1 and KV7.1/KCNE1, we anticipate overall similar endocannabinoid interactions with and without KCNE1. The preserved ARA-S and LIN-S effect upon KCNE1 co-expression is likely due to the low apparent pKa value of these compounds, which makes them more resistant to altered local pH. In this way, endocannabinoids with low enough apparent pKa can evade the indirect KCNE1-induced protonation that has been previously shown to impair the effect of PUFAs with carboxylic head groups upon KCNE1 co-expression.,The shift in V22 depletion. This indicates that endocannabinoids either do not target this site or have no functional effect at the site. This is different from the hKV7.1/KCNE1 channel modulator CP1, which can substitute for PIP2 to mediate electromechanical VSD-PD coupling.2 depletion upon incomplete depletion , suggesting that ARA-S can augment hKV7.1/KCNE1 channel function at intermediate PIP2 levels when there are some functional channels available for ARA-S to act on. In our hands, AEA and 2-AG did not have effects in the hKV7.1/KCNE1 channel, likely because they do not have a negatively charged head group. This is in contrast to TRPV1, which AEA and 2-AG directly activate by binding to a tunnel formed by the S1-S4 region and the vanilloid-binding pocket located between S3-S4 of one monomer and S5-S6 of an adjacent monomer.V7.1/KCNE1 and TRPV1 highlight that endocannabinoids can have differential sites and effects on ion channels.In addition, K249 and R259 are among the residues in the simulations that in the lower leaflet establish hydrogen bonds with LIN and LIN-S. This region was also described for LIN in a previous MD simulation study but shown in experiments to not be functional for PUFAs.V4.3 and KV1.5,,V and L-type CaV channels in ventricular myocytes.V7.1/KCNE1. The ARA-S effects we observe on hKV7.1/KCNE1 in this study, with significant effects at 300 nM, occurs at similar concentrations as those previously reported for endocannabinoids on TRPV1, KV1.5, ASIC3, and KV4.3.,,,,in vivo levels. Also, our experimental setting does not capture the complex in vivo regulation of endocannabinoids, which for instance involves the endocannabinoid carrier albumin.Endocannabinoids like 2-AG and AEA affect several cardiac ion channels, such as the potassium channels KV7.1/KCNE1. There are several compounds that restore a physiological APD and QT interval in pharmacological and/or genetic experimental models of LQTS. Most of these compounds have different mechanisms of action and binding sites compared to ARA-S. For example, the mentioned CP1 compound, in contrast with ARA-S, substitutes for PIP2 after PIP2 depletion and has been suggested to bind to the PIP2 pocket to mediate VSD-PD coupling.V7.1/KCNE1 complex, in a stoichiometry dependent manner, by specifically enhancing the current of the AO state when binding to its pocket located on the intracellular side.,V7.1 antibodies increase channel open time and open probability by targeting an extracellular region close to the selectivity filter of hKV7.1/KCNE1.V7.1/KCNE1 channel modulators make a set of chemically varied modulators with APD and QT shortening effects, acting through diverse mechanisms. This could be utilized in the development of future targeted treatment of LQTS, in which the preferred mode of modulation would be guided based on the underlying cause of the disease.Recent studies have demonstrated beneficial effects in a LQTS context of modulators augmenting the activity of hKV7.1/KCNE1 channel. The pronounced activation of hKV7.1/KCNE1 and the shortening of the APD and QT interval in guinea pig hearts by ARA-S highlight ARA-S and other negatively charged endocannabinoids as putative endogenous modulators of hKV7.1/KCNE1 and as model compounds for drug development.To conclude, this study shows that specific members within the endocannabinoid family target the cardiac hKAll authors read and approved the final version of the manuscript. I.H-I, M.A.S, S.L, J.N, S.I.L performed electrophysiology experiments and analysed related data. L.M.C-G, V.C, S.Y performed simulations and analysed related data. I.H-I, V.C, M.A.S, S.Y, S.Y.N, B.H.B, D.P.T, S.I.L contributed to study design. All authors contributed to manuscript writing. I.H-I and S.I.L verified the underlying data.sara.liin@liu.se).All numerical data of the work are provided in the main figures, supplementary figures, and tables. Any additional information, including trajectories, are openly available upon request to the corresponding author  including a description of the interaction of charged lipophilic compounds with the K


Hepatocellular carcinoma (HCC) is a malignant tumor leading cancer-associated high mortality worldwide. Unfortunately, the most commonly used drug therapeutics not only lack of target ability and efficiency, but also exhibit severe systemic toxicity to normal tissues. Thus, effective and targeted nanodrug of HCC therapy is emerging as a more important issue. Here, we design and develop the novel nanomicelles, namely Mannose-polyethylene glycol 600-Nitroimidazole (Man-NIT). This micelle compound with high purity comprise two parts, which can self-assemble into nanoscale micelle. The outer shell is selected mannose as hydrophilic moiety, while the inner core is nitroimidazole as hydrophobic moiety. In the cell experiment, Man-NIT was more cellular uptake by HCCLM3 cells due to the mannose modification. Mannose as a kind of glucose transporter 1 (GLUT1) substrate, can specifically recognize and bind to over-expressed GLUT1 on carcinoma cytomembrane. The nitroimidazole moiety of Man-NIT was reduced by the over-expressed nitroreductase with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as the cofactor, resulting in transient deletion of NADPH and glutathione (GSH). The increase of reactive oxygen species (ROS) in HCCLM3 cells disturbed the balance of redox, and finally caused the death of tumor cells. Additional in vivo experiment was conducted using twenty-four male BALB/c nude mice to build the tumor model. The results showed that nanomicelles were accumulated in the liver of mice. The tumor size and pathological features were obviously improved after nanomicelles treatment. It indicates that namomicelles have a tumor inhibition effect, especially Man-NIT, which may be a potential nanodrug of chemotherapeutics for HCC therapy. GSH will be oxidized to GSSG, and GSSG can also be reduced to GSH with nicotinamide adenine dinucleotide phosphate (NADPH) participation, which makes the GSH/GSSG redox be in a relatively constant equilibrium state. It will lead to redox imbalance when the GSH generation is insufficient, too much NADPH is consumed, or GSSG cannot be reduced in time  (SIGMA-ALDRICH), HE staining solution, 4% paraformaldehyde, Silica gel plate  and 5- aminofluorescein (DiR) (Macklin) were purchased from Innochem. Sephadex LH-20 was purchased from Shanghai McLean Biochemical Technology Co., Ltd.Human HCC cell lines HCCLM3 cells were obtained from the Chinese Academy of Science ; Dulbecco Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS) were purchased from GIBCO; Penicillin-streptomycin, Trypsin, MTT, Apoptosis Kit, Reactive Oxygen Species Assay Kit, NADPMale BALB/c nude mice (4–6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. NO.: 220518514622. All mice were fed in SFP level animal room. All animal experiments were approved by animal ethics committee of Harbin Medical University (KY2019-046). Ki67 antibody (ab16667), second antibody goat anti-rabbit IgG (BA-1000) were purchased form Abcam.1H NMR spectra of the compounds were tested by Dalian University of technology.Liquid mass spectrometer UPLC-LCT Premier XE ; High performance liquid chromatography LC-20T ; Nano-ZS90 nanoparticle size and ζ-potential analyzer  ; SYNERGY microplate reader ; Rotary evaporator ; Transmission electron microscope imaging (TEM) ; Flow cytometry ; Live Cell Station ; Fluorescence microscope ; The 2.2.2.2.1.2.2.1.1.-bottomed flask. The mixture was stirred at room temperature for 30 min. The mixture was stirred at room temperature overnight after being added Tert-Butyl bromoacetate . The solvent of resulting crude mixture was removed by vacuum, dissolved in CH2Cl2 and washed with 1 M aqueous HCl. The organic layer was dried over sodium sulfate, filtered, and removed excess solvent under reduced pressure to afford the product 1.For details, refer to 1 was dissolved in TFA/CH2Cl2 (6 mL/6 mL) under an argon atmosphere in a round-bottomed flask. The mixture was stirred at room temperature overnight, upon which time the solvent was removed by vacuum. The resulting crude solid was dissolved in ether, filtered and dried at room temperature to afford the pure product 2 : HRMS (ESI) Calcd for C5H5N3O4[M + H]+ 171; found 171.Compound 2.2.1.2.1 , N-hydroxysuccinimide  and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride  were dissolved in DMF (10 mL) under an argon atmosphere in a round-bottomed flask. The mixture was stirred at room temperature for 30 min. Compound 6  were added to the mixture and stirred at room temperature overnight. The resulting crude mixture was washed with 1 M aqueous HCl. The organic layer was dried over sodium sulfate, filtered, and removed excess solvent under reduced pressure. The crude product was purified by flash column chromatography on silica gel (MeOH/CH2Cl2 1:10) to afford the pure product 3 : HRMS (ESI) Calcd for C(9 + 2n)H(13 + 4n)N7O(4+n) [M + H]+ 724.4, 768.4, 812.4 et al.; found 724.4, 768.4, 812.4 et al.Compound 2.2.1.3.3  was dissolved in tert-butanol (10 mL) in a round-bottomed flask. A solution of compound 7  in H2O (10 mL), 0.05 M aqueous CuSO4  and 0.05 M aqueous sodium ascorbate  were added. The mixture was stirred at room temperature overnight. The solvent of crude mixture was removed by vacuum, dissolved in CH2Cl2 and then filtered the insoluble impurities. The crude product was purified by flash column chromatography on silica gel (MeOH/CH2Cl2 1:1 0 ∼ 1:5) to afford the pure product 4 (Man-NIT).Compound 2.2.1.4.2 , mPEG600  1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride , 4-dimethylaminopyridine  were dissolved in DMF (10 mL) under an argon atmosphere in a round-bottomed flask. The mixture was stirred overnight. The resulting crude mixture was washed with 1 M aqueous HCl. The organic layer was dried over sodium sulfate, filtered, and removed excess solvent under reduced pressure. The crude product was purified by flash column chromatography on silica gel (MeOH/CH2Cl2 1:10) to afford the pure product 5 (PEG-NIT).Compound 2.2.2.2.2.2.1.Man-NIT and PEG-NIT micelles were prepared at room temperature, and they were diluted to 1.0 mg/mL by deionized water. The average particle size, polydispersity index (PDI), ζ-potential were detected by Malvem Zetasizer Nano ZS 90 . TEM images of nanomicelles were obtained using a Tecnai G2 20 S-TWIN instrument. Man-NIT and PEG-NIT micelles were diluted to 0.5 mg/mL by deionized water and the ultraviolet/fluorescence absorption spectra were detected using SYNERGY microplate reader.2.2.2.2.Man-NIT and PEG-NIT micelles were stored at 4 °C for 30 days, and sampled in 0, 5, 10, 15, 20, 25, 30 days for evaluating the stability of nanomicelles by measuring the particle size and PDI. Moreover, the nanomicelles were diluted 10, 50, 100, and 500 times by deionized water, respectively. The dilution stability was evaluated by measuring the particle size and PDI of nanomicelles.2.2.2.3.Man-NIT and PEG-NIT micelles (1.0 mg/mL) were diluted to 500, 250, 125, 62.5, 31.25 μg/mL for sample solution preparation. A red blood cell suspension (2% v/v) was obtained and added to the sample solution. The mixture was gently mixed and incubated at 37 °C for 1 h. After centrifugation at 2500 rpm for 10 min, the supernate (100 μL) was taken and added to 96-well plates. At last, the absorbance was detected at 540 nm. Distilled water and normal saline mixed with 2% red blood cell suspension were used as positive control (100%) and negative control (0%), respectively. Hemolysis ratio% = (Asample-Anegative)/(Apositive-Anegative)×100%.2.2.3.2.2.3.1.5 cells/mL and continued to incubate with 20 μL Man-NIT@DiO and PEG-NIT@DiO for 4 h. Cells were washed with PBS twice and digested 3–4 min with 0.25% trypsin. The cells were centrifuged at 2000 rpm for 3 min to remove supernatant. Cell precipitation was collected and resuspended in 0.5 mL PBS. Cell suspension was measured using flow cytometry with FITC channel.DiO were encapsulated with nanomicelles to prepare the Man-NIT@DiO, PEG-NIT@DiO (1 mg/mL). HCCLM3 cells were cultured in 12-well plates overnight with a concentration of 1 × 102.2.3.2.4 cells/mL in 96-well plates for about 24 h. Then, cells were incubated with Man-NIT and PEG-NIT micelles with different concentration for 24 h. After 24 h, 100 μL fresh culture medium and 10 μL MTT (5 mg/mL) were added for another 4 h. MTT-containing solution was removed and 100 μL of DMSO was added to dissolve the MTT formazan crystals. At last, the mixture was detected at 490 nm after vibration 10 min.HCCLM3 cells were seeded at a density of 1 × 102.2.3.3.5 cells/mL in 6-well plates and treated with Man-NIT and PEG-NIT micelles (1.0 mg/mL) for 24 h. The content of NADPH was detected in each group according to the instructions of the NADP+/NADPH detection kit.HCCLM3 cells were cultured at a density of 1 × 102.2.3.4.5 cells/mL in 6-well plates and treated with Man-NIT and PEG-NIT micelles (1.0 mg/mL) for 24 h. The cells were washed with PBS, collected after centrifugation, and the supernatant was sucked up. Three times the cell precipitation volume of protein removal reagent solution was added. The samples were then subjected to two rapid freeze-thawing processes using liquid nitrogen and 37 °C water bath. The samples were placed at 4 °C for 5 min and centrifuged at 10,000 g for 10 min. The supernatant was removed and detected at 412 nm.HCCLM3 cells were cultured at a density of 1 × 102.2.3.5.4 cells/mL in 12-well plates. The cells were treated with Man-NIT and PEG-NIT micelles  for 24 h. The cells were washed with DMEM for 3 times to remove the culture medium. DCFH-DA (10 μmol/L) was diluted to 1:1000 and added to the cells. The cells were incubated with DCFH-DA at 37 °C for 20 min. The cells were washed with DMEM to remove the DCFH-DA and photoed using confocal microscope (488 nm/525 nm).HCCLM3 cells were cultured at a density of 1 × 102.2.3.6.5 cells/mL in 12-well plates and incubated with Man-NIT and PEG-NIT micelles for 4 h. The cells were washed and collected. The cells were then transferred to a 1.5 mL tube after centrifugation at 1000 rpm for 5 min. FITC-conjugated Annexin V (5 mL) and propidium iodide (PI) (10 mL) were added to incubate with the cells for 20 min at room temperature. Finally, the stained cells were measured and analyzed by flow cytometry.The percentage of apoptotic HCCLM3 cells was determined by Annexin V-FITC/PI detection kit based on the manufacturer’s protocol. In brief, HCCLM3 cells were cultured at a density of 1 × 102.2.3.7.4 cells/mL in 24-well plates and incubated with Man-NIT and PEG-NIT micelles for 4 h. Then the cells were washed with PBS and 250 μL Calcein AM/PI solution was added for incubation at 37 °C for 30 min. The cells were washed with PBS and observed using fluorescence microscope .HCCLM3 cells were cultured at a density of 1 × 102.2.4.2.2.4.1.DiR were encapsulated with nanomicelles to prepare the Man-NIT@DiR, PEG-NIT@DiR in NaCl (1 mg/mL). BALB/c nude mice were divided into three groups and injected 100 μL DiR, Man-NIT@DiR, and PEG-NIT@DiR by tail vein. The nanomicelles distribution in nude mice were photographed by Carestream in vivo FX Professional Imaging System equipped with an excitation-pass filter at 760 nm and emission-pass filter at 790 nm. The protocol of exposure: X-ray exposure time was 30 s. The data were evaluated and statistically analyzed using the Carestream imaging system.2.2.4.2.6 cells/mL) in logarithmic growth phase were taken and injected subcutaneously into the back of nude mice with insulin needle. After building the model for 5 days, the model mice were divided into model, Man-NIT, and PEG-NIT group. The groups were administered by NaCl, Man-NIT and PEG-NIT  through the tail vein daily, respectively. The treatment period was 21 days, and then the tumorigenesis of nude mice was observed daily. Another 6 unmodeled BALB/c nude mice were used as control.Twenty-four male BALB/c nude mice were conventionally fed in SPF sterile environment for one week. HCCLM3 cells  and short diameter (b) of the tumor were measured. The calculation formula of tumor volume is V = a × b2.2.4.4.After a series of processes including embedding, sectioning, baking, dewaxing, and hydration, the tumor tissues of nude mice were stained with hematoxylin solution and then stained with eosin. After dehydration, the tissue was mounted by neutral balsam and observed under an upright microscope.2.2.4.5.The expression of Ki67 protein in tumor tissues of nude mice was detected using immunohistochemistry, and the operation was performed according to the manufacturer’s protocols. The tumor sections were treated with dewaxing, rehydration, antigen repair and blocking. The sections were incubated with the first antibody Ki67 (1:100) 4 °C overnight. The next day, the sections were incubated with secondary antibodies for 1 h. After the sections were stained with diaminobenzidine and photoed using fluorescence microscope.3.3.1.1H NMR  δ 8.14 − 8.02 , 7.70 , 7.21 − 7.03 , 5.30 , 5.15 , 4.83 , 4.52 , 4.02 − 3.80 , 3.77 − 3.33 , 3.11 − 2.82 , 2.44 , 2.13 , 1.40 − 1.14 . HRMS (ESI) Calcd for C19 + 2n)H(29 + 4n)N7O(10+n) [M + H]+, 956.5, 1044.5, 1088.6 et al.; found 956.5, 1044.5, 1088.6  δ 7.21 , 7.14 , 5.19 , 4.38 − 4.27 , 3.87 − 3.43 , 3.33 , HRMS (ESI) Calcd for C(10 + 2n)H(15 + 4n)N3O(6+n) [M + H]+ 802.4, 846.4, 890.5 et al.; found 802.4, 846.4, 890.5 et al. : , 1088.6 . The pur5 et al. .3.2.The average particle size of Man-NIT and PEG-NIT were 34.7 ± 1.20 nm and 46.2 ± 2.56 nm. PDI of Man-NIT and PEG-NIT were 0.184 ± 0.05, 0.193 ± 0.06, respectively. The particle size distribution and ζ-potential were shown in 3.3.+/NADPH ratio. Compared with control group, the GSH contents of Man-NIT and PEG-NIT groups were decreased, and there was a significant difference, ***p < 0.001, ****p < 0.0001 (Flow cytometry showed that Man-NIT micelles were more cellular uptake than that of PEG-NIT micelles . Cell vi< 0.0001 . Compare< 0.0001 . The abo3.4.Flow cytometry showed that the apoptosis rate of HCCLM3 cells either treated with PEG-NIT or Man-NIT was 19.72% and 31.51%, as shown in 3.5.The nanomicelle distribution was observed in 3.6.After 14-day of nanomicelles treatment in nude mice, the body weight of mice in each group was shown in 3.7.HE staining showed that the lung cells of mice were evenly arranged with uniform density and no pathological cells in 4.In this experiment, the nanodrug Man-NIT micelles were firstly designed and synthesized. Man-NIT micelles not only exhibited nano-sized particle size and excellent stability, but also no toxicity and hemolysis. Mannose as a transport substrate of GLUT1, which could be used to modify the nanomicelles. We found that Man-NIT micelles could be taken more in by HCCLM3 cells after modification than that of PEG-NIT without mannose modification. The reason is that compared with normal cells, the tumor cells need significantly enhanced glucose uptake ability and metabolic activity to satisfy the purpose of rapid proliferation, namely Warburg effect (Poff et al., Man-NIT not only has the excellent performance of nanocarrier, but also exhibits the capability of antitumor. With the increase of nanomicelle concentration, Man-NIT can effectively inhibit tumor growth. The nitroimidazole of Man-NIT will deplete NADPH under the action of nitrogenase. Our results showed that the content of NADPH in Man-NIT group was obviously decreased, which was consistent with the literature (Guo et al., Imaging in vivo showed that Man-NIT could target the liver and mainly accumulated in the liver of mice, which was closely related to the high expression of GLUT1 on HCC cells (Liu et al., 5.A structurally novel self-assembled nanomicelles, namely Man-NIT, was designed and synthesized. The outer shell was mannose, which could target GLUT1 on the tumor cells. While the inner core nitroimidazole moiety of Man-NIT could react with NADPH, resulting in decrease of GSH and enhancement of ROS to disturb the balance of redox in tumor cells, and finally caused the tumor cells death. It indicates that Man-NIT micelles have the abilities of tumor inhibition, which may be a potential nanodrug of chemotherapeutics for HCC therapy.


The highest specific capacitance of 6.80 F g−1 was achieved for the GCE-NiPcMWCNTs electrode in 5 mM [Fe(CN)6]4−/3− prepared in 0.1 M PBS (pH 7). Charge transfer resistance Rct values of 0.06, 0.36, 0.61, and 1.98 kΩ were obtained for the GCE-NiPcMWCNTs in H2SO4, SAB, Na2SO4, and PBS electrolytes, respectively. Power density values, otherwise known as the “knee” frequency f°, of 21.2, 6.87, 2.22, and 1.68 Hz were also obtained for GCE-NiPcMWCNTs in H2SO4, Na2SO4, PBS, and SAB electrolytes, respectively. GCE-NiPcMWCNTs demonstrated the fastest electron transport capability and the highest power density in H2SO4 compared to the other electrolytes. Hence, GCE-NiPcMWCNTs-modified electrodes had high stability, high energy and power densities, and a large specific capacitance.The supercapacitive properties of synthesized nickel phthalocyanine multiwalled carbon nanotubes nanocomposite on a glassy carbon electrode (NiPcMWCNTs-GCE) in four different electrolytes were investigated. The successful synthesis of the NiPcMWCNTs nanocomposite was confirmed by UV/vis electrode spectroscopy, SEM, TEM, EDX, and XRD techniques. The supercapacitive behaviors of the modified electrodes were examined in PBS, H In recent years, the quest for environmentally sustainable energy sources other than crude oil has prompted an upsurge in energy and power research. Supercapacitors have recently received increased attention as a potential replacement for batteries and fuel cells due to their high power density and quick charging and discharging capabilities . SupercaAccording to the research, electrodes modified with CNT-MO nanocomposite showed a large capacitive current in several electrolytes ,3. Hence−1), environmental friendliness, and well-defined redox behavior, allowing its utilization in a wide range of applications, including supercapacitors and solar cells  solution in 0.1 M phosphate buffer solution (PBS) (pH 7) prepared from disodium hydrogen phosphate dehydrate (Na2HPO4·2H2O) and sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) were purchased as analytical grade reagents from Sigma-Aldrich . Nickel chloride, sodium acetate, sulfuric acid, sodium sulfate, methanol, and hydrazine hydrate were obtained from Glassworld Chemicals , while the hydrochloric acid (HCl) and sodium hydroxide (NaOH) used for adjusting the pH were purchased from Merck Chemicals (PTY) LTD . The experiment was carried out using distilled water throughout.Multiwalled carbon nanotubes (MWCNTs)  with 98%, potassium bromate  with JEOL2100 instrument fitted with a LaB 6 electron gun by JEOL Ltd, Tokyo, Japan, X-ray diffraction spectrophotometry (XRD) by Bruker Company, Karlsruhe, Germany, scanning electron microscopy by JEOL JSM-6610 LV, Dearborn, Peabody, MA, USA (SEM), UV–visible spectrophotometry by Agilent Technology, Cary 300 series UV−vis spectrometer, Darmstadt, Germany, and Energy diffraction X-ray (EDX) by JEOL JSM-6610 LV, Dearborn, Peabody, MA, USA (EDX). Both electrochemical and impedance spectroscopy (EIS) studies were conducted using an Autolab Potentiostat PGSTAT 302 , controlled by GPES (version 4.9) software . At the same time, the EIS data were obtained using Metrohm Autolab (FRA 32) NOVA 2.1.3 software  with a frequency range of 100 kHz–0.1 Hz.Nickel nanoparticles (NiNp) were made by completely dissolving 5.0 mL of hydrazine hydrate and 0.955 g of nickel chloride in 400.0 mL of ethylene glycol, and 4.0 mL of 1 M NaOH solution was added to the reaction mixture and swirled at 60 °C for 1 h in a closed flask. The resultant product (black) was completely rinsed with ethanol and dried for 24 h under vacuum at 35 °C .3 and 90 mL of conc. H2SO4 for 5 h at 55 °C and then 8 h at 80 °C. This mixture was washed several times with distilled water until a neutral pH was achieved. The obtained solid particles were dried at 70 °C for 24 h 4−/3− in 0.1 M PBS (pH 7). Impedance spectroscopy studies were performed in four different bromate electrolyte solutions: 0.1 M KBrO3 prepared in 0.1 M PBS (pH 7), 0.1 M KBrO3 prepared in 0.1 M sodium acetate buffer (SAB) (pH 7), 0.1 M KBrO3 prepared in 0.1 M Na2SO4 (pH 2), and 0.1 M KBrO3 prepared in 0.1 M H2SO4 (pH 1) at a constant potential (+0.2 V) and a frequency range between 100 kHz and 0.1 Hz. The bare and the modified electrodes  served as the working electrodes. The reference electrode used was Ag/AgCl in saturated KCl, while the counter electrode was platinum wire. All experiments were carried out at 25 ± 1 °C.Electrochemical characterization of the modified electrodes was performed by CV using 5 mM 3−/4− prepared in 0.1 M PBS at pH 7 (scan rate of 25 mV s−1). Individual peak separation (Ep) values of 0.39, 0.25, 0.18, 0.13, and 0.18 V were obtained for the GCE, GCE-Pc, GCE-Ni, GCE-fMWCNTs, and GCE-NiPcMWCNTs, respectively, as shown in pa/Ipc values of approximately one. This suggests that all reactions on the GCE and modified electrodes’ surfaces were reversible. The voltammogram in pa) of 457.0 μA was observed for the GCE-NiPcMWCNTs-modified electrode, which is about 18 times greater than those obtained with the GCE and other modified electrodes. This implies that the GCE-NiPcMWCNTs electrode has the fastest electron transport of all the electrodes. The high current response and enhanced electron transport of the GCE-NiPcMWCNTs-modified electrodes are attributed to the combined cooperative effect between NiNp, Pc, and fMWCNTs, which is the result of the larger surface area of Pc and fMWCNTs, as well as the strong electrical conductivity of the fMWCNTs and nickel. 3−/4− solution prepared in 0.1 M PBS at pH 7. CV was utilized to investigate the effect of scan rate  in 5 mM [Fe(CN)6]3−/4− solution produced in 0.1 M PBS at pH 7, the stability of GCE-NiPcMWCNTs was investigated. A slight current drop (4.3%) was exhibited by the GCE-NiPcMWCNTs-modified electrode, as shown in With 20 repeated scans (25 mVs3 solution in 0.1 M PBS (pH 7), 0.1 M KBrO3 solution in 0.1 M sodium acetate buffer SAB (pH 7), 0.1 M KBrO3 solution in 0.1 M Na2SO4 (pH 2), and 0.1 M KBrO3 solution in 0.1 M H2SO4 (pH 1). The Metrohm Autolab NOVA 2.1.3 software  was used to fit all impedance data.To extensively study both the electron transport and capacitive properties of the GCE and modified electrodes, an EIS experiment was conducted in four different electrolytes using an Autolab Potentiostat PGSTAT 302 at a frequency range of 100 kHz–0.1 Hz and a fixed potential of 0.2 V versus Ag/AgCl, saturated with 3 M KCl. The highest capacitance was found at a voltage of 0.2 V; thus, that was used for this experiment. The various electrolytes used for this study include: 0.1 M KBrO3 solution produced in 0.1 M PBS (pH 7) electrolytes. ct denotes charge-transfer resistance, C denotes capacitance, CPE denotes constant phase element, and W denotes Warburg impedance. The summary of the fitted impedance data with their chi-square x2 values is shown in 2 values and the low percentage errors (in brackets) indicate that the EIS data were successfully fitted. The fitted impedance data in ct value of 1.98 kΩ, suggesting a fast electron transfer of the GCE-NiPcMWCNTs electrode compared to the other electrodes. The high conductivity of the functionalized MWCNTs, acting as a good electron conductor between NiNp and Pc and the electrode surface, accounts for the GCE-NiPcMWCNTs’ fast electron transport. According to the results, electron transport was significantly faster at the GCE-NiPcMWCNTs electrode, aligning with CV results. This also contributed to the high Cp value of the GCE-NiPcMWCNTs electrode. The n values ranging from 0.6–0.8 for all electrodes may be attributable to the ease with which ions diffuse to and from the electrode/solution contact.Further investigation on the capacitive behaviors of the electrodes was carried out using the “knee” frequency (f°). The frequency at which capacitive behavior is prominent is referred to as the “knee” frequency. It is a measure of a supercapacitor’s power capability, as it is the transition point between the high-frequency and low-frequency components. The greater the f°, the faster the supercapacitor can charge and discharge, or the higher the power density the supercapacitor can attain ,35. The 3 prepared in 0.1 M H2SO4 (pH 1) electrolyte, with ct values obtained for the electrodes in H2SO4 electrolyte are in increasing order of 0.06 < 2.4 < 6.6 < 7.7 < 13.4 KΩ for GCE-NiPcMWCNTs, GCE-fMWCNTs, GCE-Pc, GCE-Ni, and GCE, respectively, with GCE-NiPcMWCNTs exhibiting the lowest Rct value, suggesting its fastest electron transport ability compared to the other electrodes in the electrolyte. From this result, the GCE-NiPcMWCNTs electrode exhibited faster electron transport in H2SO4 than other electrodes, similar to what was obtained for PBS. f° values of 5.18, 1.27, 2.95, 9.10, and 21.2 Hz were obtained in H2SO4 electrolyte for the GCE, GCE-Pc, GCE-Ni, GCE-fMWCNTs, and GCE-NiPcMWCNTs, respectively, with the GCE-NiPcMWCNTs electrode having the highest f° value (21.2 Hz), thus suggesting a higher power capability of the electrode.3 prepared in 0.1 Na2SO4 (pH 2) electrolyte, with 2SO4 electrolyte is not very different from the two previous electrolytes, with the GCE-NiPcMWCNTs electrode exhibiting the fastest electron transport ability. The Rct values obtained in Na2SO4 electrolyte were 0.61, 0.69, 7.1, 10.1, and 17.3 kΩ for GCE-NiPcMWCNTs, GCE-fMWCNTs, GCE-Ni, GCE-Pc, and GCE, respectively, with GCE-NiPcMWCNTs demonstrating their usual faster electron transport compared to the other electrodes in the electrolyte. An f° value of 6.87 Hz was obtained for the GCE-NiPcMWCNTs electrode in Na2SO4 electrolyte. This value is higher than the values for other electrodes, as expected, which is indicative of its higher power capability.3 prepared in 0.1 SAB (pH 7) electrolyte, with ct value of 0.36 kΩ. Additionally, a similar trend was observed in their f° values. The f° values obtained in SAB were in increasing order of GCE (0.54) = GCE-fMWCNTs (0.54) < GCE-Pc (0.95) < GCE-Ni (1.27) < GCE-NiPcMWCNTs (1.68). The GCE-NiPcMWCNTs electrode demonstrated a greater f° value (1.68 Hz) compared to the other modified electrodes, which is nearly three times greater than that of the unmodified GCE.Expectedly, the GCE-NiPcMWCNTs electrode exhibited the fastest electron transport behavior in SAB electrolyte with a lower Rct values of 1.98, 0.06, 0.61, and 0.36 kΩ were obtained for the GCE-NiPcMWCNTs electrode in PBS, H2SO4, Na2SO4, and SAB electrolyte, respectively. GCE-NiPcMWCNTs had the lowest Rct value (0.06 kΩ) in H2SO4 electrolyte compared with the other electrolytes. This shows that the fastest electron transport capability of GCE-NiPcMWCNTs was achieved in H2SO4 electrolyte, and the slowest was obtained in PBS. A comparative summary  of the E2SO4, Na2SO4, and SAB electrolyte were 2.22, 21.2, 6.87, and 1.68 Hz, respectively. The f° value for H2SO4 (21.2 Hz) is about three times higher than that for Na2SO4 (6.87 Hz). SAB had the lowest f° value  vs. log f° at −1.0 Hz, as illustrated in 2SO4 electrolyte compared to the other electrolytes. The values of the phase angles of GCE-NiPcMWCNTs in all the electrolytes were less than the anticipated 90° for an ideal capacitive property. This is a further confirmation of the GCE-NiPcMWCNTs as a non-ideal capacitor.Phase angles of ~74.0°, 42.0°, 36.0°, and 33.3° were obtained for the GCE-NiPcMWCNTs electrode in SAB, H2SO4, Na2SO4, PBS, and SAB electrolytes, respectively. These values are less than the expected slope of 1.0 for pure capacitive behaviors, affirming that the GCE-NiPcMWCNTs electrode investigated in this research is a high-specific-capacitance, high-energy-density hybrid supercapacitor that combines the faradaic pseudocapacitive and electric double-layer system.Similarly, slope values of −0.2, −0.3, −3.2, and −5.3 were obtained from the graphs of log |Z| vs. log f° for H2SO4 than the other electrodes. A similar trend in power density was observed for the GCE-NiPcMWCNTs, which had the highest power density, decreasing in the order of H2SO4 > Na2SO4 > PBS > SAB. The hybrid supercapacitor also exhibited high electrochemical stability with insignificant changes (4.3%) over 20 repeated scan rates. Hence, GCE-NiPcMWCNTs-modified electrode exhibited high energy and power densities and high specific capacitance. The enhanced electron transport capability and capacitive properties of the GCE-NiPcMWCNTs in H2SO4 compared to the other electrolytes were credited to relatively fewer H+ ions in the acid, allowing for easier ion penetration in the acidic electrolyte. The improved capacitive behavior of NiPcMWCNTs was also credited to the greater surface area of the fMWCNTs and phthalocyanine and the high electrical conductivity of the nickel and fMWCNTs. Finally, the findings of this study show that GCE-NiPcMWCNTs is a suitable electrochemical sensor for detecting bromate, especially in H2SO4 and Na2SO4 electrolyte solutions.This study demonstrated the successful fabrication and characterization of a hybrid supercapacitor, as confirmed by UV/vis spectroscopy, XRD, EDX, SEM, and TEM techniques. CV and EIS studies showed that the GCE-NiPcMWCNTs-modified electrode exhibited rapid electron transfer ability and higher supercapacitive behavior in H


The tumor suppressor p53 functions as a pioneer transcription factor that binds a nucleosomal target DNA sequence. However, the mechanism by which p53 binds to its target DNA in the nucleosome remains elusive. Here we report the cryo-electron microscopy structures of the p53 DNA-binding domain and the full-length p53 protein complexed with a nucleosome containing the 20 base-pair target DNA sequence of p53 (p53BS). In the p53-nucleosome structures, the p53 DNA-binding domain forms a tetramer and specifically binds to the p53BS DNA, located near the entry/exit region of the nucleosome. The nucleosomal position of the p53BS DNA is within the genomic p21 promoter region. The p53 binding peels the DNA from the histone surface, and drastically changes the DNA path around the p53BS on the nucleosome. The C-terminal domain of p53 also binds to the DNA around the center and linker DNA regions of the nucleosome, as revealed by hydroxyl radical footprinting. These results provide important structural information for understanding the mechanism by which p53 binds the nucleosome and changes the chromatin structure for gene activation. Significance StatementIn response to DNA damage, the transcription factor p53 stimulates the expression of various genes involving tumor suppressors by binding to the p53 binding DNA sequence, which is frequently embedded within the nucleosome. Here, we present cryo-EM structures of the p53-nucleosome complexes. In the structures, p53 peels the nucleosomal DNA region containing the p53 binding DNA sequence from the histone core, and specifically binds the DNA as a tetramer. Biochemical analyses revealed that the flexible p53 C-terminal domain, which contains an additional DNA-binding domain, binds around the center and linker DNAs of the nucleosome. These results provide novel insights for understanding the mechanism by which p53, as a pioneer transcription factor, recognizes its binding sequence in chromatin.The p53 protein is the product of a tumor suppressor gene, and functions as a transcription factor that induces the expression of genes related to cell cycle, apoptosis, senescence, and DNA repair . The humIn eukaryotes, genomic DNA is highly compacted as chromatin. The basic chromatin unit is a nucleosome, in which approximately 150 base pairs (bps) of DNA are bound to a histone octamer containing two each of histones H2A, H2B, H3, and H4 , 7. In fThe p53 tumor suppressor is a pioneer transcription factor , and binIn the present study, we reconstituted the nucleosome containing the p53BS DNA in the position found in the native nucleosome at the p21 promoter region, and determined the cryo-electron microscopy (cryo-EM) structures of the p53-nucleosome complexes. Deletion analyses of p53 domains coupled with hydroxyl radical footprinting revealed the nucleosomal DNA binding by the p53 DBD and CTD regions. These results provide structural information for understanding how p53 binds to the nucleosomal target DNA sequence in chromatin.We reconstituted the nucleosome with a 193 bp DNA containing a modified 601 sequence for the structural analysis with p53 Fig. . The recDensity corresponding to the p53 DBD was observed in approximately 70% of particles, but most of the p53 DBD map was not obvious because of the flexibility and/or multiple conformations of the p53 DBD bound to the nucleosomal DNA . We thenTo determine whether the full-length p53 (p53 FL) targets the nucleosomal p53BS DNA by the same mechanism as the p53 DBD, we purified the p53 FL protein and prepared the p53 FL-nucleosome complex . In thisIn both the p53 FL-nucleosome and p53 DBD-nucleosome structures, the p53 DBD may contact the nucleosomal histone H3 tail near the His39 residue Fig.  and B. TTo further dissect the p53 domain contribution to the nucleosome binding, we prepared a p53 mutant, p53 CTR (residues 294 to 393) containing another DNA-binding region of the p53 CTD Figs.  and S8. To clarify the interactions of the p53 regions with the nucleosomal DNA, we performed a hydroxyl radical footprinting analysis Figs.  and S9. In addition to the p53BS DNA region, the hydroxyl radical hypersensitive sites around the nucleosomal dyad are also protected by the p53 FL binding Fig. . IntriguIn the present study, we determined the cryo-EM structures of the p53-nucleosome complexes, in which the p53 DBD specifically binds to its target p53BS DNA sequence located in the natural nucleosomal position at the entry/exit region. In the structures, the p53 DBD tetramer peels the DNA from the histone surface, thus drastically changing the orientation of the linker DNA containing the p53BS DNA. Since the linker DNA orientation is a key determinant for the higher-order chromatin architecture, p53 binding to the nucleosomal entry/exit site could change the chromatin structure around its binding sites. Consistent with this idea, p53 binding to its target p53BS DNA reportedly induced changes in the local chromatin environments in cells . The p53The nucleosomal linker DNA re-orientation observed in the p53-bound state could be induced with the p53 DBD lacking the N- and C-terminal regions, indicating that the p53 DBD alone is sufficient for the specific DNA binding in the nucleosome. Intriguingly, the p53 mutants and splicing variants lacking the C-terminal DNA-binding region are proficient in the activation of p53-mediated genes , 23. TheThe present study also demonstrated that the C-terminal DNA binding region of p53 is involved in its nucleosome binding. Mice lacking the p53 CTD reportedly exhibit enhanced senescence and apoptosis . The splThe human histones were bacterially produced and purified by the method described previously . Each DNm Tris-HCl (pH 7.5), 7 m guanidine hydrochloride, and 20 mm ß-mercaptoethanol. The histone octamer was then refolded by dialysis against refolding buffer, containing 10 mm Tris-HCl (pH 7.5), 2 m NaCl, 1 mm EDTA, and 5 mm ß-mercaptoethanol, and purified by size exclusion chromatography on a HiLoad 16/60 Superdex 200 prep grade column (Cytiva). The eluted fractions were flash-frozen and stored at −80°C.The histone octamer was reconstituted and purified by the method previously described . The purm Tris-HCl (pH 7.5), 2 m KCl, 1 mm EDTA (pH 8.0), and 1 mm DTT. Nucleosomes were reconstituted by decreasing the KCl concentration to that in reconstitution buffer-low, containing 10 mm Tris-HCl (pH 7.5), 0.25 m KCl, 1 mm EDTA (pH 8.0), and 1 mm DTT. The nucleosomes were then purified by non-denaturing polyacrylamide gel electrophoresis using a Model 491 Prep Cell apparatus (Bio-Rad), and stored at −80°C in nucleosome-buffer, containing 20 mm Tris-HCl (pH 7.5), 1 mm DTT, and 5% glycerol.The nucleosomes were reconstituted by the salt dialysis method, as previously described . The DNAm IPTG. The proteins were purified with GS4B beads (Cytiva), and the GST-tag was removed by PreScission protease digestion. The untagged p53 protein was further purified by column chromatography with Heparin Sepharose 6 Fast Flow affinity resin (Cytiva) and HiLoad16/60 Superdex 200 prep grade resin (Cytiva), in p53-buffer containing 30 mm Tris-HCl (pH 7.5), 500 mm NaCl, and 2 mm ß-mercaptoethanol.The p53 FL, p53 DBD, and p53 CTR proteins were bacterially produced and purified as previously described . The DNAm) was mixed with either 0, 0.4, 0.6, or 0.8 µm of the p53 protein, and incubated at 25°C for 30 min in buffer containing 20 mm Tris-HCl (pH 7.5 or pH 8.0), 100 mm NaCl, 0.4 mm ß-mercaptoethanol, 0.7 mm DTT, and 0.01% NP−40. The reaction products were then fractionated by 5% non-denaturing polyacrylamide gel electrophoresis in 0.5xTBE buffer, and visualized by ethidium bromide staining or Cy5 fluorescence signal detection, using an Amersham Typhoon imager (Cytiva).The fluorescently labeled nucleosome  was mixed with the p53 DBD in a 6-fold molar ratio, in buffer containing 25 mm Tris-HCl (pH 7.5 or pH 8.0), 250 mm NaCl, 1 mm ß-mercaptoethanol, and 0.5 mm DTT, and incubated at 25°C for 30 min. The mixture was applied on the top of a 5% to 20% (w/v) sucrose gradient solution containing 10 mm HEPES-KOH pH 7.5, 20 mm NaCl, and 2 mm DTT, with an increasing concentration (0% to 2%) of paraformaldehyde (Electron Microscopy Sciences), and the samples were centrifuged at 27,000 rpm at 4°C for 16 hr, using an SW41 rotor (Beckman Coulter). After centrifugation, 400 µL aliquots of the sample were collected from the top of the gradient solution. The sample fractions were analyzed by non-denaturing polyacrylamide gel electrophoresis. The peak fractions were then collected and desalted with buffer, containing 20 mm HEPES-KOH (pH 8.0) and 2 mm TCEP (pH 8.0), using a PD-10 column (Cytiva). For the p53 FL-nucleosome complex, the 169 bp nucleosome  was mixed with p53 FL in a 4-fold molar ratio, in buffer containing 22.5 mm Tris-HCl (pH 7.5 or pH 8.0), 225 mm NaCl, 0.9 mm ß-mercaptoethanol, 0.45 mm DTT, and 0.01% NP-40, and incubated at 25°C for 30 min. The p53 FL-nucleosome complex was then fixed and purified by the same method as described above.The p53-nucleosome complexes for cryo-EM analysis were prepared by the GraFix method . For the2, respectively  were washed with ethyl acetate and glow-discharged by soft plasma ion bombardment . Aliquots (2.5 µL) of the p53-nucleosome complex or the p53 DBD-nucleosome complex were applied to the Quantifoil grids in the Vitrobot Mark IV chamber (Thermo Fisher Scientific) at 100% humidity and 16°C, and then blotted and plunged into liquid ethane. Data collections were performed on a Krios G4 cryo-transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV, using the EPU software. Digital micrographs of the p53-nucleosome complex and the p53 DBD-nucleosome complex were recorded on a K3 BioQuantum (Gatan) direct electron detector calibrated at a pixel size of 1.06 and 1.1 Å in the electron counting mode, using a slit width of 25 eV, and retaining 40 frames with total doses of 56.8 and 46 electron/Åectively .The image processing was performed with the RELION3.1 software . All movFor the model building of the free nucleosome, the cryo-EM structure of the nucleosome containing a 145 bp Widom-601 sequence (PDB ID: 7OHC)  was placFor the p53-nucleosome complexes, the free nucleosome model and the crystal structure of the p53-DNA complex (PDB ID: 3KMD)  were plam) was mixed with either p53 (2.4 µm), p53 DBD (3.6 µm), or p53 CTR (3.6 µm), and was incubated at 25ºC for 30 min. The buffer was exchanged into the reaction buffer, containing 5 mm Tris-HCl (pH 7.5), 5 mm NaCl, and 0.25 mm EDTA (pH 8.0), by four consecutive rounds of dilution and filtration using an Amicon Ultra-0.5 filter unit (MWCO 30 kDa). For the hydroxyl radical reaction, a 2.5 µL aliquot of 4 mm FeAmSO4/8 mm EDTA, 0.1 m sodium ascorbate, and 0.6% v/v H2O2 was simultaneously mixed with 50 µL of the sample, and then incubated for 2 min at room temperature. The reactions were terminated by adding a 5 μL aliquot of 100 mm thiourea and a 10 μL aliquot of 3 m sodium acetate (pH 5.2). The resulting DNA samples were purified by deproteinization and phenol/chloroform/isoamyl alcohol extraction, followed by ethanol precipitation. The DNA samples were denatured by Hi-Di™ formamide (Thermo Fisher Scientific) and separated by 8% Urea-PAGE in 1×TBE buffer. The Cy5 and 6FAM signals were detected with the Amersham Typhoon scanner (Cytiva). The gel images were analyzed with the ImageJ software (For the hydroxyl-radical footprinting experiments , the flusoftware .pgac177_Supplemental_FileClick here for additional data file.


Knotted1-like homeobox (KNOX) transcription factors are involved in plant development, playing complex roles in aerial organs. As Prunus species include important fruit tree crops of Italy, an exhaustive investigation of KNOX genes was performed using genomic and RNA-seq meta-analyses. Micropropagation is an essential technology for rootstock multiplication; hence, we investigated KNOX transcriptional behavior upon increasing 6-benzylaminopurine (BA) doses and the effects on GF677 propagules. Moreover, gene function in Prunus spp. was assessed by Gisela 6 rootstock transformation using fluorescence and peach KNOX transgenes. Based on ten Prunus spp., KNOX proteins fit into I-II-M classes named after Arabidopsis. Gene number, class member distribution, and chromosome positions were maintained, and exceptions supported the diversification of Prunus from Cerasus subgenera, and that of Armeniaca from the other sections within Prunus. Cytokinin (CK) cis-elements occurred in peach and almond KNOX promoters, suggesting a BA regulatory role in GF677 shoot multiplication as confirmed by KNOX expression variation dependent on dose, time, and interaction. The tripled BA concentration exacerbated stress, altered CK perception genes, and modified KNOX transcriptions, which are proposed to concur in in vitro anomalies. Finally, Gisela 6 transformation efficiency varied (2.6–0.6%) with the genetic construct, with 35S:GFP being more stable than 35S:KNOPE1 lines, which showed leaf modification typical of KNOX overexpression. Plant TALEs encompass the KNOTTED-like (KNOX) and BELL-like (BELL) factors that form heterodimers and cooperate in organ development  as query and “RNA”, “Illumina”, “paired”, and “fastq” keywords. The results were visualized in the “SRA Run Selector” tool, and runs from “aerial organs” were selected, giving priority to those with replicates (if any). Overall, 63 paired-end runs were retrieved (http://github.com/ncbi/sra-tools (accessed on 27 December 2022)) and used in PRUNOX expression meta-analyses. Raw reads were quality-checked and filtered using Trimmomatic v0.39 [KNOX expression averages  were converted into z-scores and visualized using gplots package v. 3.1.3 (https://cran.r-project.org/web/packages/gplots/index.html (accessed on 27 December 2022)).As for RNA-seq meta-analyses, publicly available transcriptomic data of etrieved  using thic v0.39 ; StringTic v0.39  was usedP. persica × P. amygdalus) were produced by CREA-OFA [shooting media SM 0, 1X, and 3X. The pH was adjusted (5.6) before agar addition, and sterilization followed at 121 °C for 20 min. All chemicals were by Sigma-Aldrich, Milan, Italy unless differently reported. Growth conditions were as follows: 24 ± 2 °C, light/dark—16/8 h, light intensity 37.5 μmol m−2 s−1 . The experimental matrix system consisted of propagules  grown on SM0 (control), 1X, and 3X and replicated to allow sampling after 24 and 72 h and to avoid “open and close” repetitions. Materials for gene expression and histologic analyses derived from the same jars. As for RNA, propagules (n ≥ 12) for each treatment and timing were subdivided into three bulks , collected in tubes, flash-frozen in liquid nitrogen, and stored at −80 °C.Micropropagated clones of GF677 rootstock ). Student’s t-test was used to assess significant differences between treated and untreated samples at a given concentration.Phenotyping was carried out at 10 days post-treatment by photographing 20 propagules per treatment and timing (5 p/jar) and measuring stem height, number of leaves on the main axis, and number of side shoots using ImageJ software (n ≥ 8) for each treatment and timing were immersed in ethanol (70% v/v) and stored at 4 °C. The dehydrated samples were embedded in Technovit 7100 , longitudinally sectioned at 8 μm with a Microm HM 350 SV microtome , dried overnight at 40 °C, stained with 1% toluidine blue (w/v), dried and permanently mounted in Canada Balsam, and observed under light microscopy. Sections were digitally photographed using a Leica DMRB optical microscope equipped with a Leica DC 500 camera.Propagules (20/random hexamer primer mixture (1:1). The cDNA (100 ng) was amplified in a volume (10 mL) containing Titan HotTaq EvaGreen qPCR Mix 1X  and 0.3 mM of each primer. Triplicate reactions were performed at the following conditions: 15 min at 95 °C followed by 40 cycles of three-step amplification . Primers are listed in PpRPII [−ΔΔCt method [Total RNA was isolated , DNAse-tn PpRPII ,69 and ct method .www.battistinivivai.com (accessed on 27 December 2022)) in vitro clones of Gisela 6 plants. The rootstock is a hybrid derived from P. cerasus L. cv. Schattenmorelle (4n = 32) × P. canescens Bois (2n = 16), ploidy 2n = 3X = 24. Apices from these clones were further subcultured on several media, and the best one—ensuring the culture of vital clones for 40 days—was QBLI (3.3 g/L), which consisted of QL medium [−1 m−2 PAR, 25 °C, and 60% HR). In vivo adaptation included clones (5 leaves and well-formed roots) that were transferred from jars to pots  and kept for 2 days in baby rooms . Two weeks later, plantlets  were moved to larger pots  in a glasshouse  under natural light photoperiod and intensity.We purchased  and RM2 (WPM + BA 2 mg/L + IBA 1 mg/L + sucrose 3% v/w) were at pH 4.8. Explants derived from shoots (length > 1 cm) from clones propagated on QLBI for 3–4 weeks. Leaves (length = 1.5–2 cm) were devoid of petioles and cut with three incisions perpendicular to the main vein . To test regeneration frequency, 50 explants (ca. 5 per Petri dish) were used for each treatment. The RM1/RM1 combination was satisfactory and used for transformation experiments.Regeneration tests were as follows: Leaf explants from healthy in vitro clones underwent a 24 h dark pretreatment in liquid medium by gentle shaking and then were transferred to agarized regeneration media (RM) under light. The combinations dark–liquid/light–solid were on RM1/RM1 and RM2/RM2 media; RM2 was recommended for Gisela 6 . RM1  and synthetizing the GFP, were used in transformation tests. The 35S:pat/35S:KNOPE1 cassettes of the binary vector pBA002, conferring resistance to phosphinothricin (PPT), were previously described [A. tumefaciens strain EHA105 by freeze–thaw method.The escribed . All recA. tumefaciens (At) were grown  in LB (10 mL) plus antibiotics ; then, they were collected , suspended in RM1 (25 mL) supplied with acetosyringone (100 µM), and brought to 0.5 value of OD600. The explants were incubated in bacterial suspension  for 3 days, and then At was removed by washing in RM1  plus cefotaxime (500 mg/L) 6 times, followed by 5 washes with RM1 to remove the antibiotic. The explants were dried on sterile filter paper and were laid on solid RM1 plus cefotaxime (250 mg/L) in Petri dishes  for a fortnight at 25 °C. Afterward, the explants were moved onto solid RM1 without cefotaxime and with PPT (20 mg/L) or HYG (10 mg/L). PPT and HYG concentrations were established by previous dosage assays. Putative transgenic clones were screened based on PPT or HYG resistance (absence of chlorosis/necrosis) combined with a phenotypic trait (fringed leaves due to KNOPE1 or GFP fluorescence), moved to fresh media containing PPT or HYG for further selection for ca. 2 weeks, and finally transferred to rooting medium (without herbicide or antibiotic).Single colonies of recombinant HPTII probe (ca. 1100 bp) was excised  from pCAMBIA1302; the BAR probe (ca. 676 bp) was excised  from pBA-KNOPE1 plasmid [Genome insertion was checked by Southern blot  and PCR  plasmid . Fragmen2 (2.5 mM), 1.25 U Taq polymerase, and 1X Buffer (Dream Taq Fermentas). Cycle conditions were as follows: denaturation start (95 °C/5 min), 35 amplification cycles , and final extension (72 °C/5 min). Primers are listed in As for PCR, the reaction mixture final conditions in 30 µL were as follows: gDNA (300 ng), primers (0.3 μM), dNTPs (0.2 mM), MgClKNOPE1-like messages were not detected or strongly downregulated in leaf blades (devoid of petioles); the use of specific primers allowed the detection of peach KNOPE1 expression from the transgene 35S:KNOPE1 by RT-PCR in clones previously checked for the transgene in the genome. The cDNA synthesis was described above; the PCR conditions (in 50 µL) were as follows: 200 ng cDNA, 1 µM of each primer, 0.5 mM dNTPs, 2.5U DreamTaq , 1X buffer; start cycle 95 °C/5 min, 35 cycles: 95 °C/1 min, 62 °C/30 s, and 72 °C/90 s, final extension: 72 °C/5 min; the products (15 µL) were electrophoresed (0.8% agarose gels). Primers are listed in 35S:GFP Gisela 6 was detected using an Eclipse 80i microscope  equipped with FITC filters (B-1E) and NIS-Element BR 2.20 software (Nikon) by directly observing organ tissues of transgenic and control plants.In Gisela 6 nontransformed plants, the t-test and two-way ANOVA. Pearson correlations were calculated using the “rcorr” function in the R environment v3.4.3 ).Data were analyzed by different statistical tests including Student’s