When tested in combination with a Na metal anode and a non-aqueous NaClO4-based electrolyte solution in coin cell configuration, the CuMgTi-571-based positive electrode enables an 87% capacity retention after 500 cycles at 120 mA g−1 and about 75% capacity retention after 2000 cycles at 1.2 A g−1.P2-type sodium manganese-rich layered oxides are promising cathode candidates for sodium-based batteries because of their appealing cost-effective and capacity features. However, the structural distortion and cationic rearrangement induced by irreversible phase transition and anionic redox reaction at high cell voltage  cause sluggish Na-ion kinetics and severe capacity decay. To circumvent these issues, here, we report a strategy to develop P2-type layered cathodes via configurational entropy and ion-diffusion structural tuning. In situ synchrotron X-ray diffraction combined with electrochemical kinetic tests and microstructural characterizations reveal that the entropy-tuned Na The use of Mn-rich layered cathodes in Na-based batteries is hindered by inadequate cycling reversibility and sluggish anionic redox kinetics. Here, the authors report a strategy to stabilize the structure and promote anionic redox via configurational entropy and ion-diffusion structural tuning. The foremost challenge in advancing NIBs lies in developing high-performance and low-cost electrode materials. Among the available cathode materials, P2-type layered oxides, especially sodium manganese-rich oxides (NaxMnO2) have been deemed as one of the most fascinating cathode candidates due to their high specific capacities and the natural abundance of Mn6. Moreover, oxygen anionic redox activity has been confirmed in these cathodes when charging beyond 4.0 V (vs. Na/Na+), which can increase the reversible capacity to even over 200 mAh g−1 at low charge/discharge specific current 8. However, compared with Li+, the insertion/extraction of Na+ in the reported P2 cathodes generally shows poor reversibility and sluggish kinetics owing to its larger ionic radius  have attracted significant interest as a low-cost alternative to Li-ion systems for large-scale energy storage applications due to the natural abundance of Na resources and the similar chemical properties of Na and Li14. However, the reported cycle stability by surface coating or aliovalent doping, especially under fast-charge conditions remain far less satisfied19. Instead, rather less attention has been paid to improving ion transport kinetics. Ion transport kinetics within electrode materials play a pivotal role in the battery performance, which depends highly on the host structure and ion-diffusion tunnels. A stable host structure can avoid the collapse of lattice and diffusion channels, which is the precondition for reversible ion insertion/extraction. The construction of more diffusion-favorable tunnels is beneficial for the rapid migration of large amounts of ions. Thus, the key to addressing the slow ion kinetics lies in constructing a stable host structure with more migration tunnels.Over the past decades, extensive research has been conducted to stabilize anionic redox in the Mn-based P2-type layered cathodes21. Hu et al. has revealed that high-entropy O3-type NaNi0.12Cu0.12Mg0.12Fe0.15Co0.15Mn0.1Ti0.1Sn0.1Sb0.04O2 cathode could enable reversible O3-P3 phase transition and cationic redox within a narrower voltage range of 2.0–3.9 V20. In addition to structural stability, {010} planes with an open structure and ion-migration tunnels are favorable active planes for ion transport of layered cathode materials. Our previous work has demonstrated that increasing {010} active planes could significantly improve the rate performance of lithium layered oxide cathodes23. P2 cathodes with increased exposure of {010} facets have also been reported, which however were evaluated only within a low-voltage region (2.0–4.0 V).Recently, high-entropy oxides have shown significantly improved structural stability during charge/dischargeHigh-voltage operation (>4.2 V) is an effective way to increase the specific capacity of battery materials. However, it is well-known in the battery community that high-voltage operation often leads to undesired structural degradation of the cathode. To date, despite numerous attempts through various strategies, the stabilization of layered cathodes at high voltage remains a formidable challenge. To the best of our knowledge, the correlation between entropy and anionic redox kinetics/reversibility of layered cathodes at high voltage remains elusive.0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2  layered cathodes with different configurational entropy and {010} active facets. Through systematically comparing their electrochemical performance and investigating their phase transition and Na+ diffusion coefficient during charge/discharge, we have identified the correlation between entropy and active crystal facet, cationic/anionic kinetics as well as an electrochemical performance of P2-type cathodes. In comparison with Na0.62Mn0.67Ni0.37O2, the optimal Na0.62Mn0.67Ni0.23Cu0.05Mg0.07Ti0.01O2 cathode with higher entropy and increased active {010} facet not only effectively suppressed destructive structural variation and extends cycle life, but also significantly promoted the anionic redox and fast charging capability during high-voltage cycling .Based on the above discussions, simultaneously increasing the entropy and active facets would be a powerful gateway to enable P2-type layered cathodes with ultrafast charging capability and ultralong cycle life Fig. , which h0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 and the non-entropy-tuned NaMNO2 were characterized by X-ray diffraction (XRD) together with the Rietveld refinement. As shown in Fig. 25. The pure P2 phase of Na0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 suggests uniform incorporation of Cu2+, Mg2+, and Ti4+ into the lattice of the materials. Compared to Mn4+ (0.53 Å) and Ni2+ (0.69 Å), the introduction of larger-size cations such as Cu2+ (0.73 Å) and Mg2+ (0.72 Å) may lead to a debate on the size variation of interlayer spacing. However, previous literature has shown that interlayer spacing doesn’t always increase with an increased substitution amount of large-sized cations16. Meanwhile, a high entropy sample Na0.62Mn0.67Ni0.23Co0.033Al0.033Ti0.02O2 consisting of small-sized cations  was synthesized and showed that introduction of cations with smaller ionic radii into P2-typed structure can lead to increased interlayer spacing . The interlayer spacings (do-o) of CuMgTi-533, CuMgTi-552, CuMgTi-571, and NaMNO2 were 4.09, 4.03, 4.09, and 3.98 Å, respectively. The maximum changes of interlayer distance (d) are 0.11 Å. Compared to previous report work by increasing the Na+ layer distance from 3.6 to 5.8 Å to improve the electrochemical performance26, the interlayer distance change here is much smaller.Therefore, to obtain the lattice parameters and interlayer spacing of Na0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 and the non-entropy-tuned NaMNO2 were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Low-magnification SEM images , as displayed in Supplementary Table 0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2, and Na, Mn, Ni in the NaMNO2 samples.The morphologies and detailed crystal structures of the entropy-tuned Na+ transport between the MO2 layers    patterns of CuMgTi-571 Fig.  exhibiteers Fig. . Surpris9%) Fig. . The incSconfig) of Na0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 materials depends on the number and mole fraction of the elements, which can be calculated according to Eq.  and γ0 are surface energy at any temperature and 0 K, respectively. T is temperature, A is Helmholtz free energy, Svib and Sconfig are the system’s vibrational and configurational entropy, respectively. It can be found that the surface energy of a system with simple composition and low entropy is mainly influenced by temperature, while the surface energy of polycomponent system with high entropy is mainly influenced by configurational entropy Sconfig28. The P2-type layered cathode materials in our current work contain six cations, which are high-entropy systems. The surface energy of these P2-type entropy-tuned materials is mainly affected by configurational entropy Sconfig at a given temperature. Thus, configurational entropy variation may reasonably alter the surface energy of the crystal plane, influencing the growth rate of different facets and promoting the formation of the layered cathode with more {010} facets. Therefore, high configurational entropy is the impetus for the growth of a high percentage {010} active facets in this work, while the temperature is the driving force for the growth of {010} active facets in the case of O3 lithium compounds (LiNi1/3Co1/3Mn1/3O2 and Li(Li0.17Ni0.25Mn0.58)O2) with low entropy23. These results indicate that  entropy variation may reasonably alter the surface energy of crystal planes influencing the growth rate of different facets29. To the best of our knowledge, the effect of configurational entropy on the surface structure of electrode materials has been barely reported and requires more comprehensive study.Based on the calculation formula of surface energy (Eq. ):2\docum0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 and the non-entropy-tuned NaMNO2. As shown in Supplementary Fig. 0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 and NaMNO2 cathodes exhibit multiple pairs of redox peaks, corresponding to a series of phase transition reactions occurring during charge/discharge. According to the previously reported results, the low-voltage reduction peak located at ca. 2.26 V is associated with the reduction reaction of Mn4+/Mn3+  analysis  (1 ≤ n ≤ 3) are the redox-active couples within the potential range applied and will contribute to the capacity of the as-prepared cathodes.To clearly unravel the effect of entropy and crystal-facet modulation on the structural stability and performance of P2-structured cathode material, a series of electrochemical characterizations were performed. Cyclic voltammetry (CV) measurements were conducted to compare the redox behaviors of the entropy-tuned Na2−/O2n−) (1 ≤ n ≤ 3) is reversible in the first CV curves of CuMgTi-571 and NaMNO2  of anionic redox reaction in NaMNO2 is 0.59 V . In contrast, the (ΔEP) of anionic redox reaction in CuMgTi-571 is decreased to 0.33V .The anionic redox reaction (O2 at 0.1 C (12 mA g−1) of different cycles , respectively. It can be seen that the NaMNO2 undergoes a severe voltage fade and further specific capacity decay during cycling, which are characteristic features of Li-excess cathodes that also involve oxygen anionic redox38. By contrast, the CuMgTi-571 cathode exhibits less voltage fading during the cycling test in comparison with the NaMNO2 cathode, indicating that the voltage fade issue in CuMgTi-571 is alleviated. This result can be seen more clearly in the normalized voltage capacity curve in Supplementary Fig. 2 with much lower entropy at 0.1 C (12 mA g−1). As shown, CuMgTi-571 could deliver an initial discharge capacity of 148.2 mAh g−1, and can still maintain 132.9 mAh g−1 after 100 cycles, resulting in capacity retention of 89.6%. Although NaMNO2 delivers a comparable first discharge capacity (150.5 mAh g−1) with CuMgTi-571, the discharge capacity of NaMNO2 decreases fast and remains only 60.2% (90.6 mAh g−1) of its initial capacity after 50 cycles. Moreover, the average coulombic efficiency of CuMgTi-571 achieves 98.0% compared to 95.4% of NaMNO2, suggesting the fast dynamics and high reversibility of CuMgTi-571. To investigate the reversibility of electrochemical reaction in CuMgTi-571 and NaMNO2, dQ/dV curves after 50 cycles at 0.1 C (12 mA g−1) rate are displayed in Supplementary Fig. 2. These sharp contrasts confirmed that entropy modulation can significantly stabilize the structures and anionic redox of P2 layered cathodes during high-voltage charge/discharge.Figure 0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 and the non-entropy-tuned NaMNO2 were further evaluated at various charge/discharge rates from 0.1 C to 10 C , the CuMgTi-571 cathode enables cell-specific capacities of 143.5, 128.3, 122.9, 120.1, 115.1, 104.4, and 82.6 mAh g−1, respectively. When the C-rate switches back to 0.1 C (12 mA g−1), a discharge capacity of 151.8 mAh g−1 can be immediately recovered, indicating the tolerance for fast Na+ transport and stable structure of CuMgTi-571 cathode. The changes in the interlayer distance (d) between NaNMO2 and CuMgTi-571 are only 0.11 Å. Such a small variation of interlayer distance is not sufficient to account for the improvement in the rate capability. In contrast, the relatively larger entropy difference  and active-facet percentage difference  should play a dominant role in the observed improvement on rate capability. With an increase in the entropy and active facet, the reversible capacities of Na0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 under various rates were increased accordingly.The rate capability of the entropy-tuned Na−1) for 500 cycles  rate, respectively. CuMgTi-571 cathode again displays less voltage fading during the long cycling compared with the NaMNO2 cathode. Figure 0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 and NaMNO2 at 1 C (120 mA g−1) for 500 cycles. It can be clearly seen that the CuMgTi-571 cathode with the highest entropy and {010} active facet percentage also displays better long-term stability with the highest reversible capacity (103.3 mAh g−1) and capacity retention (87%) after 500 cycles. While the reversible capacities and retention of CuMgTi-552  and CuMgTi-533  were decreased accordingly. In sharp contrast, the NaMNO2 with the lowest entropy and least {010} active facet exhibits rapid capacity decay, which can only maintain a reversible capacity of 30.9 mAh g−1 after 500 cycles. In addition, the NaMNO2 cathode also exhibits a lower Coulombic efficiency than the CuMgTi-571 cathode during charge/discharge . The discharge capacity of the NaMNO2 cathode faded rapidly during cycling and dropped to 22.9 mAh g−1 at the 1000th cycle , Supplementary Fig. To further verify the benefit of entropy-driven structural stability on the Na+ diffusion coefficients (DNa+) of CuMgTi-571 and NaMNO2 cathode were measured through the galvanostatic intermittent titration technique (GITT), and the detailed calculation process is described in Supplementary Note DNa+ of CuMgTi-571 and NaMNO2 in four voltage regions. The average DNa+ for both samples are around 10−8 cm2 s−1 in the low and middle voltage region (2.0–4.05 V) during either charging or discharging, where the charge compensation is mainly contributed by the cationic redox . In contrast, the average DNa+ for both samples are around 10−11 cm2 s−1 in the high-voltage range of 4.05–4.3 V, where the charge compensation is mainly contributed by the anionic redox (O2−/O2n−) (1 ≤ n ≤ 3). The results indicate that anionic redox has much slower kinetics than cationic redox, which is the rate-determining step. Thus, enhancing the kinetics of anionic redox is crucial for the performance improvement of Mn-based P2-type layered cathodes. From Fig. DNa+ of CuMgTi-571 at high voltage is higher than those of NaMNO2. During 4.05–4.3 V, the average DNa+ of CuMgTi-571 is ~10 times higher than that for NaMNO2, suggesting the kinetics of anionic redox is greatly improved in the entropy and crystal-facet tuned cathode.The Na2 at high voltage, in situ electrochemical impedance spectroscopy (EIS) experiments were conducted using Na metal as a counter/reference electrode. Supplementary Fig. 2 at different potentials of the first charge and discharge process. Upon Na+ insertion/extraction, both samples experience a series of processes in the bulk and at the surface, leading to different suppressed semicircles in Nyquist plots of the first charge and discharge process39. At the high voltage region (4.0–4.3 V), the Nyquist plots of both samples exhibit two distinct parts including a depressed semicircle in the high-frequency region and an inclined line in the low-frequency zone. The high-frequency semicircle should be related to the charge-transfer resistance41. By comparing the diameters of the semicircles in the high-voltage region  ions, which could cause phase transition and new phase formation. To check the impact of entropy and crystal-facet tuning on the structural stability of the material, in situ synchrotron high energy X-ray diffraction (HEXRD) was conducted on CuMgTi-571 during charge/discharge , and the results are displayed in Fig. −1) between 2.0 and 4.3 V, respectively. Upon Na+ extraction (charge), the (002) and (004) peaks shift toward a lower 2θ° angle due to the increase in the repulsive electrostatic interaction between adjacent MO2 sheets; while (100), (102), (108), and (112) peaks consecutively peaks shift toward a higher 2θ° angle with the contraction of a-, b-axis44. During the subsequent discharge, the XRD pattern of CuMgTi-571 exhibited an exactly opposite evolution. During the whole charge/discharge process, the well-defined peaks corresponding to the P2 phase are well maintained and returned to the initial states after one full cycle. No new peaks belonging to OP445, O28, or Z6 phase are detected when charged to 4.3 V, and no existing peaks vanish or split, indicating that new phase formation and phase transition do not occur in this material. The changes in lattice parameters upon cycling based on the refinement results are displayed in Fig. a, c, and V in the first cycle are only 0.74, 0.91, and 0.57%, respectively, which are much smaller than those of the state-of-the-art reported layered cathodes (Supplementary Table + (de-)intercalation manifests the unusual structure stability of entropy and crystal facet tuned cathode.Nas; while 0, (102),2 materials were scraped from 4.2 V-charged cathodes. The (002) peak, which is most sensitive to the Na content during charging/discharging, is usually used to monitor the phase transformation of P2-type layered cathodes during heating. The (002) reflection of charged CuMgTi-571 continuously moves to a lower angle with increasing the temperature due to the lattice expansion caused by heating  peak fading starts at 350 and 322 °C for the CuMgTi-571 and NaMNO2 cathodes, respectively, further confirming the inferior structural stability of the NaMNO2. The formation of a new phase and inferior structural stability would lead to irregular migration channels and hence sluggish Na+ diffusion.In situ HEXRD during heating of de-sodiated cathode powder was also performed to investigate the phase stability of entropy and crystal-facet tuned cathodes at high temperatures (heating from 30 to 450 °C) to investigate their thermal tolerance Fig. . The tesing Fig. . Distinc °C Fig. . Such ph2 after 500 cycles at 1 C (1 C = 120 mA g−1) were further compared . SEM images in Fig. +. In contrast, the morphology of the NaMNO2 particles changed after long-term cycling intercalation. Moreover, the increased {010} electrochemically active planes due to the increased entropy create more favorable and efficient tunnels for the migration of Na+, which facilitate rapid Na+ (de-)intercalation and promote electrochemical kinetics.The above results clearly demonstrate that fast and reversible (de-)intercalation of large Na+ kinetics, and electrochemical performance of the Na0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 was systematically studied by a combination of TEM, electrochemical measurement, in situ XRD, in situ EIS and GITT. The results demonstrate that increased entropy and active facet can effectively stabilize crystal structure and accelerate electrochemical kinetics of sluggish anionic redox, thus enabling the fast and reversible migration of Na+. In virtue of entropy-driven structural stability and increased migration channels, an optimal CuMgTi-571 cathode with higher entropy and a more active facet shows fast charging/discharging capability up to 10 C (~6 min) and long-term durability for over 2000 cycles.In summary, configurational entropy could result in a thermodynamically stable structure through a local minimization of Gibbs free energy, and a high percentage of {010} facets in the layered cathodes could provide more channels for ion transport. Based on the advantages of high entropy and high active facets, a combinatorial strategy of entropy modulation and active-facet modulation was developed to stabilize the structure and optimize ion-migration pathways of P2-type layered cathodes. The correlation of entropy, active facet, Na0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2, y = 0.01, 0.02, and 0.03) were prepared using a stirring hydrothermal method. In a typical synthesis, stoichiometric amounts (with 7% excess sodium) of NaCH3COO·3H2O, Mn(CH3COO)2·4H2O, Ni(CH3COO)2·4H2O, Cu(CH3COO)2·H2O, Mg(CH3COO)2·4H2O, and TiO2 were mixed in 60 mL of deionized water. An appropriate amount of H2C2O4  was used as a precipitant agent and also dissolved in deionized water. H2C2O4 solution was added dropwise into the above mixture under stirring. Afterward, the reaction mixture was put into 100 mL Teflon-lined autoclaves and aged at 130 °C for 12 h under stirring. The green precipitates were collected by solvent evaporation at 80 °C. The resulting precipitates were annealed in air at 500 °C for 6 h and then at 900 °C for 12 h, and P2-Na0.62Mn0.67Ni0.23Cu0.05Mg0.09-2yTiyO2 samples were finally obtained. In addition, Na0.62Mn0.67Ni0.37O2, Na0.62Mn0.67Ni0.21Cu0.05Mg0.016Zn0.016Sn0.016Y0.016O2, and Na0.62Mn0.67Ni0.21Cu0.05Mg0.015Zn0.015Sn0.015Zr0.015O2 were also prepared by same method. The obtained P2-type layered Na0.62Mn0.67Ni0.23Cu0.05Mg0.03Ti0.03O2, Na0.62Mn0.67Ni0.23Cu0.05Mg0.05Ti0.02O2, Na0.62Mn0.67Ni0.23Cu0.05Mg0.07Ti0.01O2, and Na0.62Mn0.67Ni0.37O2 samples were labeled as CuMgTi-533, CuMgTi-552, CuMgTi-571, and NaMNO2, respectively.A series of multi-element P2-structured layered oxides , transmission electron microscopy , inductively coupled plasma-mass spectrometry , X-ray diffraction , energy dispersive X-ray , and X-ray photoelectron spectroscopy .2O < 0.1 ppm, O2 < 0.1 ppm). The electrodes were rinsed using dimethyl carbonate  repeatedly, then dried in glovebox to remove the solvents. For ex situ XPS measurements, the dried electrodes were first placed into a vacuum transfer vessel in glovebox and then transferred into the XPS ultrahigh vacuum chamber. For ex situ XRD measurements, the dried electrodes were transferred from glovebox and then tested in air.The cycled electrode samples for ex situ XPS and XRD characterization were obtained by disassembling the cycled half-cells using Na metal anode in Ar-filled glovebox  dissolved in propylene carbonate  with 2 vol% fluoroethylene carbonate . A constant charge/discharge current of 20 mA g−1 was applied by using a MACCOR system between 2.0 and 4.3 V at 25 °C. Each 2D diffraction pattern was obtained by a PerkinElmer amorphous silicon 2D detector during the charge/discharge process and then were converted to 1D patterns using fit2D software with a calibrated using a standard CeO2 sample with the same experimental setup. The postmortem cathode HEXRD measurements during the heating was also conducted at 11-ID-C of Advanced Photon Source. Both the NaNMO2 and NiMgTi-571 cathode were charged to 4.2 V and then disassembled in the glovebox and scratch off the active materials carefully. The charged cathode materials were then loaded into a stainless-steel DSC autoclave and sealed in the glovebox to prevent any humid/air contamination during the in situ heating experiment. A Linkam TS1500 furnace was used to heat the charged samples from 30 to 450 °C at 5 °C min−1 and a PerkinElmer 2D detector was continuously used to acquire the 2D diffraction data during the heating process.The in situ synchrotron high energy XRD characterizations of the CuMgTi-571 electrode were carried out at the beamline 11-ID-C of the Advanced Photon Source at Argonne National Laboratory with a wavelength of 0.1173 Å. The Na|CuMgTi-571 coin cells (type CR2032) with two identical holes on the top and bottom cases were assembled in the argon-filled glovebox and then sealed with Kapton tape after filling with the electrolyte. The electrolyte was 1 M NaPFN-methyl-2-pyrrolidone  on an Al foil  and drying the slurry under vacuum at 120 °C for 12 h. The areal loading of the active material was about 3–4 mg cm−2. A high-purity Na metal foil was used as anode and glass fiber as separator . Na metal foil  was made from Na chunks under mineral oil, which was removed from the surface of Na by dried paper inside the glovebox. Na chunk was then pressed into a thin sheet under 50 Pa inside the glovebox, which was further punched into 16 mm diameter plates and used as an anode. The electrolyte was 1 M NaClO4 dissolved in a mixed solvent of propylene carbonate (PC) and fluoroethylene carbonate (FEC) . The amount of electrolyte used in the coin cells was controlled at around 280 µL. Charge-discharge experiments were performed galvanostatically between 2.0 and 4.3 V at different C-rates (1 C = 120 mA g−1) on a LANHE-CT2001A test system . Both in situ XRD cells were charged and discharge under 2.0–4.3 V. Cyclic voltammetry (CV) measurements were carried out on a CHI760E electrochemical workstation  at a scanning rate of 0.2 mV s−1 within the potential of 2.0–4.3 V. Galvanostatic intermittent titration technique (GITT) tests were conducted in the voltage range of 2.0–4.3 V by applying repeated current pulses at a current rate of 0.1 C for 15 min followed by a 3 h rest. The in situ electrochemical impedance spectroscopy (EIS) experiments were executed using a PARSTAT2273 electrochemical workstation in a frequency range from 0.1 Hz to 100 KHz with an AC amplitude of 5 mV. EIS tests of Na||CuMgTi-571 and Na||NaMNO2 coin cells were performed after each potentiostatic intermittent titration technique (PITT) program completed. The step potential difference was 0.1 V, and the step elapsed time was 3600 s. Each EIS test contains 50 data points. All the electrochemical tests were made at 25 °C.All the electrochemical measurements were performed using CR2016 coin cells assembled in an Ar-filled glovebox. Cathodes were prepared by casting a slurry composed of 70 wt% active material, 20 wt% acetylene black, and 10 wt% polyvinylidene difluoride  binder dissolved in Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Mycobacterium tuberculosis (M. tb) cell wall, and is essential for pathogen virulence and intracellular growth. Multiple proteins  in the SL-1 synthesis pathway can be treated as drug targets, but, to date, their structures have not been solved. The crystal structures of FadD23 bound to ATP or hexadecanoyl adenylate was determined in this study. We have also investigated long-chain saturated fatty acids as biological substrates of FadD23 through structural, biological, and chemical analyses. The mutation at the active site of FadD23 greatly influences enzymatic activity. Meanwhile, the FadD23 N-terminal domain alone cannot bind palmitic acid without C-terminal domain facilitation since it is almost inactive after removing the C-terminal domain. FadD23 is the first protein in the SL-1 synthesis pathway whose structure has been solved. These results reveal the importance of the C-terminal domain in the catalytic mechanism.Sulfolipid-1 (SL-1) is located in the  Sulfolipid-1 (SL-1), one of the important unique components of the pathogenic M. tuberculosis cell wall  is a communicable disease that is a major cause of ill health and death worldwide . Mycobacell wall , is a keell wall . In addiell wall . Collect659 (659 to form SL1278 (1278 with two multiple methyl-branched (hydroxy) phthioceranoic acids, most likely through membrane-associated Chp1 to produce fully elaborated SL-1  disaccharide with four acyl groups : a strai659 . Thereafm SL1278 . Additioted SL-1 . These mted SL-1 . In addited SL-1 .M. tuberculosis  tag at the N-terminus. The resulting plasmid was transformed into E. coli BL21(DE3) plySs. Cells were grown in Lysogeny broth (LB) medium with 100 μg/ml ampicillin at 37°C. When the optical density at 600 nm (OD600) reached 0.7, the culture was treated with 0.2 mM isopropyl-D-thiogalactopyranoside (IPTG) at 37°C to induce protein expression. To induce FadD23 expression in Mycobacterium smegmatis (M. smeg) mc2155 cells, fadD23 was sub-cloned into a pMV261 vector with an 8 × His tag at the C-terminus, under the control of an acetamide promoter. The resultant plasmid was transformed into M. smegmatis mc2155 cells by electroporation. The cells were cultivated in LB liquid media supplemented with 50 μg/ml kanamycin and 20 μg/ml carbenicillin. When the cells were grown to an optical density (OD600) of 1.0–1.2 at 37°C, overexpression of the recombinant protein was induced by 0.2% (w/v) acetamide at 16°C. The Escherichia coli and M. smegmatis mc2155 cells were then harvested within 5 and 96 h of induction, respectively, and resuspended in lysis buffer . Thereafter, cell debris was removed by high-speed centrifugation at 18,000 rpm for 40 min after crushing using a homogenizer . Next, the supernatant was loaded onto an Ni-NTA affinity column  and washed in the resuspension buffer . Subsequently, the protein was eluted in elution buffer . FadD23 was further purified by ion-exchange and size-exclusion chromatography , 14% (w/v) polyethylene glycol 3,350, and 9% (w/v) polyethylene glycol 4,000. The crystal of the AMP-PNP-FadD23 complex suitable for diffraction experiments contained 70 mM Tris (pH 8.5), 140 mM sodium citrate tribasic dihydrate, 140 mM ammonium phosphate monobasic, 35% w/v (+/−)-2-methyl-2,4-pentanediol, and 14% (w/v) polyethylene glycol 3,350. The crystal of the FadD23 N-terminal domain suitable for diffraction experiments contained 70 mM Bis-Tris pH 6.5, 60 mM sodium chloride, 20% (w/v) polyethylene glycol 3,350, and 6% (w/v) polyethylene glycol 6,000. Single crystals were carefully harvested from clustered crystals and were soaked in a well solution supplemented with 20% glycol for 10 s before been frozen in liquid nitrogen.Apo-FadD23 only yielded poor-quality crystals that did not produce usable diffraction data. Holo-FadD23 produced crystals suitable for diffraction experiments. 1 μl FadD23 (8 mg/ml), containing 2 mM ATP and 2 mM MgCl2.3.M. tb FadD28 [RCSB Protein Data Bank code (PDB: 3E53)] N-terminal as a search template. The output model of the molecular replacement subsequently built the C-terminal using electron density and was subjected to iterative cycles of manual model adjustment using Coot  at 100 K. Data integration and scaling were performed using XDSgui . The string Coot . Refinem2.4.The full AlphaFold v2.0 (AlphaFold2) pipeline was obtained from DeepMind and installed onto a local workstation . Structu2.5.via an electrospray ionization source mass spectra were recorded in the negative ionization mode over a 50–1,200 m/z mass range. The scan parameters included 2.5 kV capillary voltages and 1 s scan times. Other source conditions were as follows: 100°C source temperature, 50 and 600 l/h cone, and desolvation gas flow, respectively, and 40 V cone voltage. Data processing and analysis were performed using MassLynx V4.1 .The mass spectrometer (MS) analysis samples were: 200 μl of purified FadD23 (1 mg/ml) or buffer extract fatty acid using 600 μl of 100% trichloromethane. Then, the solution was dried by Termovap Sample Concentrator , and dissolve in 100% acetonitrile. The MS analysis of the FadD23 sample and unknown fatty acids was performed using exact mass TOF-MS  2.6.2, and 2 mM fatty acid  dissolved in 100% Dimethyl sulfoxide (DMSO) with a final well volume of 30 μl. The solution was allowed to equilibrate at 25°C for 20 min before a thermal melt protocol was initiated using a Bio-Rad CFX96 RT-PCR instrument . The “FRET” channel was selected to satisfy the excitation and emission wavelengths of SYPRO Orange (λex 470 nm/λem 570 nm). The melt sequence involved first maintaining the sample at 20°C for 5 min before linearly increasing the temperature by 1°C/min with fluorescence measurements every 30 s. Data were plotted as the derivative of the melting curve as a function of temperature.The Differential scanning fluorimetry DSF method is a widely used for determining protein stability. When a ligand is bound to the protein, the stability of the protein increases, such that the exposed hydrophobic groups at the same temperature decrease; a higher temperature is required to fully expose the hydrophobic groups of the protein. In a 96-well Real-time Quantitative PCR (RT-PCR) plate, a solution was prepared with a final concentration of 1 mg/ml FadD23 protein, 9× SYPRO Orange Protein Gel Stain , 2 mM ATP, 2 mM MgCl2.7.FadD23 was immobilized on a Series S Sensor Chip CM5  to 3,000 response units (RUs) using a Biacore X100  and running buffer composed of 10 mM PBS pH 7.4, 0.05% Tween 20, and 5% DMSO. Serial dilutions of fatty acid were injected at concentrations ranging from 100 to 0.19 mM. Response curves were fit to a 1:1 binding model using Biacore X100 Evaluation Software .2.8.2, 1 mM DTT, 150 mM hydroxamate (pH 7.0), 0.1 U nucleoside phosphorylase (PNP), 0.04 U pyrophosphatase, 0.2 mM MesG, 1 mM ATP, 7.5 μM FadD23, and varying concentrations of fatty acids. Reactions were run in a 96-well plate  and monitored at A360 using a Varioskan™ LUX multimode microplate reader . The kinetic parameter of the substrate was determined for FadD23 using the hydroxamate formation assay; the standard assay conditions, described above, were used. The initial rate was measured as a function of the substrate concentration to provide a saturation curve, which was fit by nonlinear regression analysis to the Michaelis–Menten equation using GraphPad Prism 8.4. Then, kcat was determined kcat = Vmax/[Et]. All mutations or truncations determine initial rate under sufficient and consistent substrate conditions (100 μM).The 100-μl reaction system contained 20 mM Tris pH 8.0, 2 mM MgCl3.3.1.M. tb FadD23 proteins failed to grow suitable crystals for data collection. However, well-shaped crystals from M. tb FadD23 with AMP-PNP or ATP could be diffracted to resolutions of 2.25 and 2.68 Å for data collection, respectively  and a small C-terminal domain (473–584), connect by flexible loops and an anti-parallel β-sheet (465–472), was obtained. Mycobacterium tuberculosis FadD23 has 14 helices, 23 β-sheets, and some irregular loops  near ATP or AMP-PNP ; the statistics and final refinement results are shown in It was observed that two  AMP-PNP . After c AMP-PNP , it was  AMP-PNP . We surm3.2.M. tb FadD23 hydrophobic pocket. Since M. tb FadD23 can obtain saturated fatty acids from the expression system, it is speculated that the protein can bind fatty acids. Therefore, the affinity of fatty acids of different lengths was determined to infer the range of carbon chain lengths with better binding capacity.We suggested that palmitic acid in the structure was bound in the 2+. After data processing, we observed that adding palmitic acid to FadD23 slightly increased the melting temperatures , indicating enhanced protein stability . The affinity of ATP to FadD23 reached 1 μM, and palmitic acid to FadD23 reached 3.450 × 10−5 M . At the M. tb FadD23 under different substrate conditions. The Km, kcat, and kcat/Km value for lauric acid are 33.89 μM, 2.40 × 10−3 s−1, and 71.8 s−1 mol−1 L, respectively. The Km, kcat, and kcat/Km value for palmitic acid is 14.26 μM, 6.72 × 10−4 s−1, and 49.4 s−1 mol−1 L, respectively. However, the Km value could not be fitted well with octanoic or stearic acid as substrates. It worth noting that the lower Km value was that palmitic acid as substrate, a higher kcat/Km value was obtained with lauric acid as substrate  FadD32, M. smeg FadD32, and M. tb FadD10  was conserved in FAALs. The backbone oxygen of Ala328 (M. tb FadD23) interacted with N37 of adenosine, Nα of Ala332 interacted with AMP, and the hydrogen bond between the OD1 atom of Asp446 (M. tb FadD23) and O38 of the ribose group were also observed in other FadDs .When  220 kDa . After v4.M. tb FadD23 can transfer hexadecanoyl adenylate or AMP-C18 to Pks2 in vivo  to elucidate the conformation changes of  process . Because process . FadD23 g FadD32 , and is eraction . We confM. tb FadD28 or PhU-AMS for M. tb FadD32, could be treated as general inhibitors for different FAALs. In vitro anti-tubercular activity experiments proved that PhU-AMS inhibits M. tuberculosis with a minimum inhibitory concentration 3.13 μM . As M. tb FadD23 and M. tb FadD28 have 66% sequence similarity and share the same evolutionary branch, it is very interesting that PhU-AMS have no effect on enzymatic activity of M. tb FadD23. Therefore, we speculate that PhU-AMS has no inhibitory effect on FadD23 because it considerably differs from its optimal substrate as the FAAL class of FadDs appears to functional nonredundant in production defer fatty acids , accession numbers 8HD4, 8HDF, 8HCZ.The data presented in the study are deposited in the RCSB Protein Data Bank  and Z.R. conceived the study. M.Y., W.Z. (Wei Zhang), L.C., L.Z. performed the experiments. M.Y., L.C., W.Z. (WeiHong Zhou) and W.Z. (Wei Zhang) analyzed the data. M.Y., X.L. and W.Z. (Wei Zhang) wrote the paper. W.Z. (Wei Zhang) and Z.R. supervised this project.This work was supported by the National Key Research and Development Program of China (Grant No. 2017YFC0840300), the National Natural Science Foundation of China , and the Fundamental Research Funds for the Central Universities, Nankai University .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Neo-vascularization is a potential target to image tumor microenvironment, as it defines its brain invasion. We investigate [18F]rhPSMA-7.3, [18F]FET, and [18F]fluciclovine were i.v. injected with 10.5 ± 3.1 MBq, 8.0 ± 2.2 MBq, 11.5 ± 1.9 MBq  and up to 90 min PET/MR imaged 21/28 days after surgery. Regions of interest were delineated on T2-weighted MRI for (i) tumor, (ii) brain, and (iii) the inferior vena cava. Time-activity curves were expressed as SUV mean, SUVR and PKM performed using 1-/2-tissue-compartment models , Patlak and Logan analysis (LA). Immunofluorescent staining (IFS), western blotting, and autoradiography of tumor tissue were performed for result validation.[18F]rhPSMA-7.3 showed a tumor uptake with a tumor-to-background-ratio (TBR) = 2.1–2.5, in 15–60 min. PKM (2TCM) confirmed higher K1  and volume of distribution VT  in the tumor region compared to the brain. Linearity in LA and similar k3 = 0.6 and k4 = 0.47  indicated reversible binding. K1, an indicator for vascularization, increased . IFS confirmed co-expression of PSMA and tumor vascularization. [18F]fluciclovine showed higher TBR  and VS  compared to [18F]FET and LA indicated reversible binding. VT increased  for [18F]FET (0.5–1.4) and [18F]fluciclovine (0.84–1.5).[18F]rhPSMA-7.3 showed to be a potential candidate to investigate the tumor microenvironment of GBM. Following PKM, this uptake was associated with tumor vascularization. In contrast to what is known from PSMA-PET in prostate cancer, reversible binding was found for [18F]rhPSMA-7.3 in GBM, contradicting cellular trapping. Finally, [18F]fluciclovine was superior to [18F]FET rendering it more suitable for PET imaging of GBM.[The online version contains supplementary material available at 10.1007/s00259-022-06040-z. The aggressive and lethal brain tumor, glioblastoma multiforme (GBM), is the most frequent form of glioma with an incidence of 3 per 100,000 in the USA  and the 18F]fluciclovine, based on a synthetic leucine-derivate [18F]FET [11C]-methionine based on its low uptake in healthy brain tissue [18F]fluciclovine does not show radio metabolites nor incorporation into proteins and hence facilitates the data interpretation with regard to PKM FET . Furthern tissue . Differed to PKM –13.The increased radiolabeled amino acid uptake is not directly correlated to higher protein synthesis of the growing tumor or to blood–brain barrier (BBB) breakdown but rather a measure of increased L-amino acid transporter (LAT) and alanine-serine-cysteine-transporter 2 (ASCT2) expression in tumor vasculature . Hence, 18F]rhPSMA-7.3 was originally developed for prostate cancer (PC) diagnostic imaging. Furthermore, it was recently investigated in a clinical study of PC with PET showing high uptake and favorable kinetics compared to current standard methods [18F]rhPSMA-7.3 has not been considered for PET imaging approaches involving GBM.A novel class of so called radiohybrid PSMA (rhPSMA) ligands is emerging . The rad methods . So far,18F]rhPSMA-7.3 for imaging of GBM related neo-vasculature in an orthotopic mouse model of GBM after 21 and 28 days of tumor growth in terms of kinetic uptake and binding characteristics. Furthermore, PET imaging of amino acid transporters was conducted to investigate its contribution to tumor vasculature/progression using PKM and the results compared to [18F]rhPSMA-7.3. Subsequently, possible advantages of the designated orphan drug [18F]fluciclovine over the commonly [18F]FET for GBM PET imaging were tested in the same setting confirming its suitability for diagnostic GBM PET.In the present study, we investigated the utility of rhPSMA-7.3, n = 5, 21 days; n = 6, 28 days), 11.5 ± 1.9 MBq , and 8.0 ± 2.2 MBq , and PET acquisition started for 90 min ([18F]rhPSMA-7.3: 60 min).Simultaneous dynamic PET and MRI was performed using a dedicated 3-ring PET insert with 150 mm field of view for whole-body mouse imaging, depth of interaction crystal matrix, and 0.7 mm spatial resolution . The micThe list-mode data were reconstructed into 24 × 5 s, 8 × 60 s, (10) × 300 s or (16) × 300 s, 60 min, or 90 min time frames, respectively, using 3D ordered subset expectation maximization with 1 iteration, 32 subsets, and a VOXEL size of 0.42 mm, applying correction for random coincidences, decay, deadtime, and scatter correction.For validation of the PET image-based results autoradiography, immunofluorescent staining and western blotting was performed as described in the Supplemental Information S2.4, S2.6, and S2.7. Written patient consent was obtained and approved by a local ethics committee .3) (MTV50) for [18F]FET/[18F]fluciclovine [18F]rhPSMA-7.3, a threshold of 75% (MTV75) was used  on the PET data by applying a threshold of 50% of the maximum ROI value 18F]rhPSMA-7.3, a plasma-to-blood ratio of 1.66, was chosen from the literature [18F]rhPSMA-7.3 and radio-TLC are presented in the supplemental information Fig. [terature  and conf(ii)18F]fluciclovine is metabolically very stable, and no radiometabolite correction was applied [[ applied –13. Furt applied .(iii)18F]FET, the plasma-to-blood ratio  was chosen from the literature  and fitted in PMOD to correct the whole blood curve. Although high metabolic stability [18F]FET and radio-TLC are presented in the supplemental information Fig. fluciclovine in the other hemisphere was lower  compared to [18F]FET   between [18F]FET and [18F]fluciclovine uptake in the tumor region  Fig. . SimilarFET Fig.  resultin01) Fig. . Only fluciclovine into the tumor cell (via ASCT2/LAT1) was significantly higher . VT (1.5/0.5) and Vs (1.3/0.3) were significantly higher in the tumor region compared to the healthy brain (p << 0.001). For both, k3 and VT, parametric maps  meaning the ratio of radiotracer in the tissue and the total parent fraction in plasma, which was confirmed by the parametric maps , k3 , and k2 .Comparing [TBR Fig.  which is18F]rhPSMA-7.3 confirmed tumor uptake and low uptake in the healthy brain  and VS (p < 0.05) for [18F]fluciclovine than for [18F]FET which is a substrate for LAT2 only. Although both amino acid radiotracers express similar SUVmean, the TBR of [18F]fluciclovine is higher due to the lower uptake in normal brain tissue. While LAT transports large neutral amino acids into the brain, ASCT transfers non-essential neutral small amino acids into the endothelial cells [+-depended amino acid transporters help to maintain the neutral amino acid concentration to a certain level [18F]FET PET presented marked uptake of the radiotracer into the GBM at 28 days with high TBR and increasing SUVmean over time, as shown for other preclinical models FEl models . The visn et al. VT, the perfusion independent concentration of the amino acid radiotracers, is significantly increasing indicating higher amino acid consumption and LAT/ASCT expression [18F]FET, K1 is decreasing which is in line with findings for necrotic tumor areas FET. Additionally, Zagzag et al. [VT for [18F]rhPSMA-7.3 at 28 days where angiogenesis and thus increased neo-vascular PSMA expression take place. McKelvey et al. [VT which might be related to binding of [18F]rhPSMA-7.3 to neo-vascularization. Concurrently, increased cell proliferation is maintained which is in line with increased VT of [18F]FET/[18F]fluciclovine FET as major model assumptions are violated: (i) Reference and target region should be represented by a 1TCM and (ii) the distribution volume is the same for target and reference region rhPSMA-7.3 and [18F]FET. (iii) The idIF was derived from the inferior vena cava, and among the scientific literature, there is some controversy about the ideal model [18F]FDG studies, the left ventricle was not visible in the PET images; thus, vena cava provided the most reliable data, as shown by others too rhPSMA-7.3, first time successfully applied in GBM PET, revealed visible tumor uptake. Following PKM, we suggest that this uptake is associated to neo-vascularization, while the number of mature vessels decreases, leading to necrosis. Imaging tumor angiogenesis using [18F]rhPSMA-7.3 with respect to tumor grading might be an interesting approach for future diagnostic PET in brain cancer. However, further research is needed to explain the mechanism behind its uptake, with special regard to the role of endothelial cells and the internalization of [18F]rhPSMA-7.3. Furthermore, the diagnostic efficacy of [18F]fluciclovine was investigated and compared to [18F]FET. Resultantly, [18F]fluciclovine showed more suitable imaging properties over [18F]FET based on higher TBR. Results from PKM confirmed higher rate constants and macro parameters when applying [18F]fluciclovine, rendering it more suitable for in vivo PET imaging of GBM.The investigation of [Supplementary file1 (PDF 6850 kb)Below is the link to the electronic supplementary material.


Toxoplasma gondii is an apicomplexan pathogen able to infect a wide range of warm-blooded animals, including humans, leading to toxoplasmosis. Current treatments for toxoplasmosis are associated with severe side-effects and a lack efficacy to eradicate chronic infection. Thus, there is an urgent need for developing novel, highly efficient agents against toxoplasmosis with low toxicity. For decades, natural products have been a useful source of novel bioactive compounds for the treatment of infectious pathogens. In the present study, we isolated eight natural products from the crude extract of the endophytic fungus Paraboeremia selaginellae obtained from the leaves of the plant Philodendron monstera. The natural products were tested for inhibiting Toxoplasma gondii proliferation, and their cytotoxicity was evaluated in different human cell lines. Six natural products showed antitoxoplasma activity with low or no cytotoxicity in human cell lines. Together, these findings indicate that biphenyl ethers, bioxanthracenes, and 5S,6S-phomalactone from P. selaginellae are potential candidates for novel anti-toxoplasma drugs. Toxoplasma gondii is an obligate intracellular protozoan parasite member of the phylum Apicomplexa, which includes known human pathogens such as Plasmodium sp., Eimeria sp., Neospora, Babesia, Theileria, and Cryptosporidium spp., with which it shares significant biological similarities + .3-(4-Hydroxy-2-methoxy-6-methylphenoxy)-5-methylbenzene-1,2-diol (B): Brown oil; UV (MeOH) λmax 211.7, 286.3 nm; 1H NMR (CDCl3), see m/z 277.1065 [M + H]+ .Cyperin (C): Brown oil; UV (MeOH) λmax 212.1, 279.8 nm; 1H NMR (CDCl3), see m/z 261.1126 [M + H]+ .ES-242-1 (D): Brown amorphous powder; [α]24D +18 ; UV (MeOH) λmax 239.0, 309.8, 345.8 nm; 1H NMR (CDCl3) and 13C NMR (CDCl3), see m/z 622.2644 [M + NH4]+ .ES-242-3 (E): Brown amorphous powder; [α]24D +66 ; UV (MeOH) λmax 239.2, 298.6, 309.4 nm; 1H NMR (CDCl3) and 13C NMR (CDCl3), see m/z 638.2588 [M + NH4]+ .Phomalactone (F): light yellowish oil; [α]24D +172 ; UV (MeOH) λmax 216.0 nm; For the details of VCD, ECD, and OR calculations, see Supplementary Materials;1H NMR (CDCl3) and 13C NMR (CDCl3), see m/z 155.0702 [M + H]+  and m/z 137.0597 [M − OH−]+ .Methyltriaceticlactone (G): White amorphous powder; UV (MeOH) λmax 290.5 nm; 1H NMR (DMSO-d6) and 13C NMR (DMSO-d6), see m/z 141.0549 [M + H]+ .S 39163/F-1 (H): Brown amorphous gum; [α]24D −11 ; UV (MeOH) λmax 218.2, 238.8, 291.9 nm; 1H NMR (CDCl3), see m/z 661.4312 [M + H]+ .A–F and pyrimethamine -2,5-diphenyltetrazolium bromide (MTT) test was used to assess cell viability of the isolated active compounds against Hs27 cells. The MTT assay is a colorimetric reaction based on the enzymatic reduction of MTT to MTT-formazan, which is catalyzed by mitochondrial succinate dehydrogenase .50 values) of the tested natural products on Hs27 cells was calculated and all data were analyzed using Prism GraphPad version 9.2.0 software.In brief, Hs27 cells were seeded 96-well plates in a monolayer in Iscove’s modified Dulbecco’s medium  and incubated at 37 °C with different concentrations of the tested natural products  in the culture media. Staurosporine , a well-known cytotoxicity-inducing kinase inhibitor , untreatStaphylococcus aureus  or Pseudomonas aeruginosa  were grown in Mueller-Hinton broth (MHB) at 37 °C shaking at 120 rpm to reach an optical density of approx. 0.4. The cell suspension was adjusted to 106 CFU/mL, of which 50 µL was seeded into a prepared 96-well polystyrene round-bottom plate containing test compounds diluted in MHB in a 1:1 serial dilution ranging from 100 µM to 0.78 µM. The plates were incubated at 37 °C statically for 24 h, and readout was performed using the BacTiter Glo assay (Promega) following the manufacturer’s instructions. Briefly, BacTiter Glo reagent was added to a white flat-bottom 96-well plate, and an equal volume of bacteria suspension was added to each well and mixed carefully. After 5 min, the luminescence was measured using a TECAN plate reader. The growth was calculated in regard to the vehicle (DMSO) and sterile control. Moxifloxacin and cefuroxime were used as a positive and negative control, respectively. All compounds were tested in triplicate.Testing for antibacterial activity was done as described previously . BrieflyM. tuberculosis H37Rv, the Minimal Inhibitory Concentration (MIC) was determined in 96-well microtiter plates containing a total volume of 100 µL employing a resazurin reduction assay [M. tuberculosis culture was pre-grown to an OD600 nm of approx. 0.4–0.6 by shaking at 37 °C in PETG square bottles  containing 10 mL supplemented 7H9 medium. The cell density was adjusted to an OD600 nm of 0.08 . Rifampicin and DMSO were used as a positive and solvent control, respectively. The 96-well plates were incubated for 5 days at 37 °C and 5% CO2 in humidified atmosphere. Afterwards, 10 µL of a 100 mg/mL resazurin solution was added to each well and resuspended carefully. After another 24 h at room temperature, the cells were fixed by adding 100 µL of a 10% formalin solution to each well. The readout was performed using a TECAN plate reader at 535 nm excitation and 590 nm emission. The growth was calculated in relation to the solvent control being 100% growth. The experiment was performed in triplicate.For the testing against on assay . Brieflyl-glutamine and supplemented with 10% fetal calf serum (FCS) and 1% sodium pyruvate. Huh-7 cells were cultured using a 1:1 mixture of RPMI 1640 medium containing 2 mM L-glutamine and 10% FCS medium and DMEM containing 10% FCS and 1% sodium pyruvate. The HEK-293 cells were cultured with DMEM including 2 mM l-glutamine and supplemented with 1% NE amino acids, 1% 1.0 mM sodium pyruvate and 10% FCS. All three cell lines were then incubated at 37 °C in an atmosphere of 5% CO2 under humid conditions for 2 weeks while renewing the medium twice weekly. Subsequently, the cells were suspended and adjusted to a density of 2 × 105 cells/mL. In a 96-well flat-bottom microtiter plate, the cells were adjusted to a total volume of 100 μL containing 2-fold serial dilutions of the tested compounds A–F ranging from 100 to 1.56 μM. Cycloheximide  was used as a positive control. After an incubation time of 48 h at 37 °C in an atmosphere of 5% CO2 under humid conditions, 10 μL resazurin solution (100 μg/mL) was added to each well and incubated for another 4 h. The fluorescence was then quantified using a Tecan Infinite 200pro microplate reader . The residual growth was calculated relative to non-inoculated conditions (0% growth) and controls treated with DMSO (100% growth).The cytotoxicity study was carried out using the THP-1 (human monocytic leukemia cell line), Huh-7 (Human liver carcinoma cell line), and HEK293 (human embryonic kidney cell line) cell lines as described before . The THP


Wistar rats were given an i.p. injection of 60 mg/kg D-gal and 10 mg/kg AlCl3 to induce AD and three doses of 1 mg/kg, 5 mg/kg or 10 mg/kg 5-MTHF by oral gavage. A positive control group was treated with 1 mg/kg donepezil by gavage. Morris water maze performance showed that 5 and 10 mg/kg 5-MTHF significantly decreased escape latency and increased the number of platform crossings and time spent in the target quadrant for AD rats. The administration of 10 mg/kg 5-MTHF decreased the brain content of amyloid β-protein 1-42 (Aβ1-42) and phosphorylated Tau protein (p-Tau) and decreased acetylcholinesterase and nitric oxide synthase activities. Superoxide dismutase activity, vascular endothelial growth factor level and glutamate concentration were increased, and malondialdehyde, endothelin-1, interleukin-6, tumor necrosis factor-alpha and nitric oxide decreased. The administration of 10 mg/kg 5-MTHF also increased the expression of disintegrin and metallopeptidase domain 10 mRNA and decreased the expression of β-site amyloid precursor protein cleavage enzyme 1 mRNA. In summary, 5-MTHF alleviates memory impairment in a D-gal- and AlCl3-exposed rat model of AD. The inhibition of Aβ1-42 and p-Tau release, reduced oxidative stress, the regulation of amyloid precursor protein processing and the release of excitatory amino acids and cytokines may be responsible.The effects of 5-methyltetrahydrofolate (5-MTHF) on a rat model of Alzheimer’s disease (AD) induced by D-galactose  and aluminum chloride (AlCl The prevalence of dementia is projected to double in Europe and triple globally by 2050, affecting 131 million people  and metaFolic acid is supplied both from the diet and through intestinal microbial synthesis. It acts as a carrier of one-carbon units during metabolic reactions and protects against neural tube defects, megaloblastic anemia and cancer . Supplem3). Learning and memory functions are investigated. Levels of Aβ1-42, phosphorylated Tau protein (p-Tau) and amino acid neurotransmitters are measured and brain acetylcholinesterase (AChE), nitric oxide synthase (NOS) activities, serum oxidative stress and cytokine indices are evaluated. Hippocampal pathological sections and APP processing are also assessed. The aim is to expose potential therapies for AD patients with disorders of folate metabolism and to generate data for drug development.Little data regarding the effects of 5-MTHF on neurological conditions is available. The potential benefits of 5-MTHF for neuropathy in depressive patients  have bee3 (99% purity), 5-MTHF (≥95% purity) and donepezil (≥98% purity) were obtained from Macklin Biochemical Co., Ltd. . Aspartic acid (Asp), glycine (Gly), glutamate (Glu) and γ-aminobutyric acid (γ-GABA) reference standards (≥98% purity by HPLC) were obtained from Solarbio Science & Technology Co., Ltd. . AChE, NOS, superoxide dismutase (SOD), nitric oxide (NO) and malondialdehyde (MDA) kits were obtained from the Nanjing Jiancheng Bioengineering Institute . Aβ1-42, p-Tau, endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) ELISA kits were acquired from Jianglai Biological Technology Co., Ltd. . Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), disintegrin and metallopeptidase domain 10 (ADAM10) and β-site amyloid precursor protein cleavage enzyme 1 (BACE1) were purchased from General Biosystems Co., Ltd. . Reverse transcription and PCR amplification kits were purchased from Accurate Biological Engineering Co., Ltd. .D-gal (98% purity), AlCl3 for 6 weeks, as described previous [3 intraperitoneally daily for 6 weeks, followed by the gavage of 1 mg/kg 5-MTHF, 5 mg/kg 5-MTHF, 10 mg/kg 5-MTHF and 1 mg/kg donepezil daily for 6 weeks, respectively [A total of 72 specific pathogen-free (SPF) male Wistar rats 150–180 g) were acquired from Sibeifu Biotechnology Co., Ltd.  in 2019-0010 and housed at 22 ± 1 °C and 60 ± 5% humidity, with a 12 h light/12 h dark cycle and access to food and water ad libitum. Animals were allowed to adapt for one week in a clean, well-ventilated animal room before the formal experiment. All animal procedures were approved by the Animal Ethics Committee of Preventive Medicine of Shandong University (ID Number: LL20200802) and were carried out following China’s Animal Care and Use Guidelines. The rats were divided into six groups (n = 12) matched by body weight after adaptive feeding: Control, AD, AD + 1 mg/kg 5-MTHF, AD + 5 mg/kg 5-MTHF, AD + 10 mg/kg 5-MTHF and AD + donepezil groups. The model of AD was established by daily intraperitoneal (i.p.) injection of 60 mg/kg D-gal and 10 mg/kg AlClprevious ,18. AD +0 g were The Morris water maze was utilized to evaluate learning and memory. It consists of a cylindrical pool with a diameter of 180 cm and a height of 80 cm, including a circular platform (diameter: 14 cm and height: 20 cm) in the second quadrant. A camera was installed above the pool to record swimming trajectories in real-time and transfer data to the operating system. The pool and surroundings were painted black, the four quadrants were indicated by space markers and light was blacked out with a curtain. The water temperature was maintained at 23–25 °C, and the platform was 1–2 cm below the water surface. Rats were put into the water from the four quadrants and the time was recorded to them finding the platform, up to a maximum of 60 s (escape latency). Animals were allowed to rest on the platform for 10 s if they found it within 60 s, but if not, the researcher would guide them to the platform to rest for 10 s and latency was recorded as 60 s. Daily latency was calculated as the mean of four trials. Rats were also put into the water from the fourth quadrant after the platform had been removed to swim for 60 s (probe task). The number of platform crossings and time spent in the target (second) quadrant were automatically recorded through the apparatus.After the water maze tests were completed, all rats were made to fast for 24 h with water ad libitum. Two rats were chosen at random from each group for cardiac perfusion and brain tissue fixed with 4% paraformaldehyde for hematoxylin–eosin (HE) staining. The remaining rats were anesthetized with an i.p. injection of 10% chloral hydrate; blood was taken from the abdominal aorta and the serum separated by centrifugation at 3500 rpm for 10 min and stored at −80 °C. Brain tissue and hippocampus were snap-frozen and stored at −80 °C.1-42 and p-Tau concentrations and AChE and NOS activities. Assays were performed using kits in accordance with the manufacturers’ instructions.Brain tissue was added to saline pre-cooled at 4 °C in a weight:volume ratio of 1:9, 1 mm enzyme-free zirconia grinding beads was added for homogenization before centrifugation at 2500 rpm for 10 min and the supernatant used for measurements of AβThen, 5% serum was used to determine SOD activity, 100% for MDA content and 100% for NO concentration; 20% serum was used to determine the VEGF, ET-1, IL-6 and TNF-α levels.Brain tissue was fixed with 4% paraformaldehyde and hippocampal tissue cut into 3 mm sections with a double-sided blade dehydrated with gradient ethanol  in an embedding box and cleaned in xylene. Tissue blocks were embedded, cut into 5 μm slices and dried on glass slides for 2 h in an oven at 60 °C. Paraffin sections were dewaxed (2 × 5 min washed with xylene), an ethanol gradient applied (100–95–85–75% for (2 min each), stained with HE for 5 min, differentiated in hydrochloric acid/ethanol for 30 s, stained with eosin for 2 min, dehydrated with 95% ethanol  and absolute ethanol , washed with xylene and sealed with neutral gum. Sections were washed with running water after hematoxylin, hydrochloric acid/ethanol and eosin application. Pathological changes to the hippocampus were observed with an inverted optical microscope .A 10% homogenate was made by adding 0.1 g brain tissue to 0.9 mL 50% acetonitrile and the supernatant used. Standard amino acid solutions of 5, 10, 50, 100, 200 and 400 μg/mL (300 μL) were measured, and the samples were mixed with 200 μL 0.5 mol/L sodium bicarbonate and 100 μL 0.5% 2,4-dinitrofluorobenzene (DNFB) and allowed to react in the dark for 55 min at 65 °C. Concentrations of Asp, Glu, Gly and γ-GABA were determined by HPLC , with separation by an Intersil ODS-3 C18 column  at 40 °C. The mobile phase was 0.05 mol/L sodium acetate  and 50% acetonitrile (solvent B), with solvent B being increased from 12% to 45% in 0.1–4.0 min, increased from 45% to 60% in 4.0–20.0 min, decreased from 60% to 12% in 20.0–31.0 min and kept at 12% for 4.0 min, at a 1.0 mL/min flow rate. A photodiode array detector and a wavelength of 360 nm were used. The injection volume was 10 μL. Amino acid standard curve parameters are shown in Total RNA was extracted from 3 samples of hippocampal tissue randomly selected from each group, and the concentration and purity were assessed. Genomic DNA was removed and RNA reverse transcribed to cDNA. GAPDH  cDNA was diluted 15-fold and ADAM10 and BACE1 cDNA 4-fold to ensure that the appropriate cycle threshold (Ct) was reached in the thermocycler. Primer sequences were as follows: GAPDH Forward: 5′ TCTCTGCTCCCCTCCCTGTTCT 3′; Reverse: 5′ ATCCGTTCACACCGACCTTC 3′; 95 bp; ADAM10 Forward: 5′ CAAAAACACCAGCGTGCCA 3′; Reverse: 5′ TCGTAGGTTGAATTGTCTTCCAT 3′; 93 bp; BACE1 Forward: 5′ GCAGTCAAGTCCATCAAGGC 3′; Reverse: 5′ GGCCGTAGGTATTGCTGAGGA 3′; 194 bp. PCR was performed as follows: preincubation at 95 °C for 30 s; PCR at 95 °C for 5 s, 60 °C for 30 s, 40 cycles; melting curve at 95 °C for 5 s, 60 °C for 1 min, increased to 95 °C; cooling at 40 °C for 15 s. Ct values were recorded, and the relative expression of target gene mRNA was calculated.p < 0.05 (two-tails). SPSS 26.0 software was used for all statistical analysis and GraphPad Prism 9.0.0 for the construction of figures.Experimental results were tested for normality by the Shapiro–Wilk method and normally distributed data expressed as mean ± SEM. Escape latency was analyzed by repeated measures ANOVA and other normally distributed and equal variance data by one-way ANOVA. The Bonferroni test was utilized for pairwise comparison. Dunnett’s T3 test was utilized to verify the difference between groups of data with unequal variance. Significant differences were defined as time = 98.31, p < 0.001; F group = 9.87, p < 0.001). A typical swimming trajectory on the fourth day of training for the six groups of rats is shown in p < 0.05), indicating successful AD modeling. Time taken to find the target platform in AD rats dosed with 1, 5 or 10 mg/kg 5-MTHF or donepezil was lower than the untreated AD group on days 3–4 , and all doses of 5-MTHF and donepezil increased these two indicators relative to untreated AD rats . Aβ1-42 increased by 77.2% and p-Tau by 92.1% (p < 0.001) in AD rats relative to controls. Dosing AD rats with 1 mg/kg 5-MTHF reduced Aβ1-42 to 64.1%, with 5 mg/kg 5-MTHF to 64.8%, with 10 mg/kg 5-MTHF to 55.6% and with donepezil to 71.9% compared with untreated AD rats (p < 0.001). Similarly, dosing AD model rats with 5 mg/kg 5-MTHF reduced p-Tau to 66.6%, with 10 mg/kg 5-MTHF to 67.6% and with donepezil to 78.1% compared with untreated model rats (p < 0.05). AChE and NOS activities in the brains of AD rats were about 2.7 and 1.3 times those in controls, respectively (p < 0.001). Supplementation with 10 mg/kg 5-MTHF reduced AChE and NOS activities to 38.4% and 82.9% in the AD model rats (p < 0.05).Differences in brain Aβp < 0.001). Treatment with 1 mg/kg 5-MTHF increased SOD by 23.5%, with 5 mg/kg 5-MTHF by 23.0%, with 10 mg/kg 5-MTHF by 30.1% and with donepezil by 36.0% compared with untreated AD rats  and was decreased by 29.2% after treatment with 5 mg/kg 5-MTHF, by 30.9% with 10 mg/kg 5-MTHF and by 38.6% with donepezil . ET-1 levels were reduced to 77.9% of those in the AD rats by 5 mg/kg 5-MTHF and to 72.1% by 10 mg/kg 5-MTHF (p < 0.01). NO levels were reduced as follows: 1 mg/kg 5-MTHF to 61.4% of values for AD rats, 5 mg/kg 5-MTHF to 56.1%, 10 mg/kg 5-MTHF to 62.2% and donepezil to 61.4% (p < 0.001). Serum TNF-α was 94.4% higher and IL-6 32.6% higher in the AD model than in controls (p < 0.001), but values were reduced as follows: 1 mg/kg 5-MTHF: TNF-α by 22.7%, IL-6 to 80.5%; 5 mg/kg 5-MTHF: TNF-α by 25.6%, IL-6 to 85.7%; 10 mg/kg 5-MTHF: TNF-α by 24.6%, IL-6 to 73.6%; donepezil: TNF-α by 22.9%, IL-6 to 81.3%  and was increased 1.7-fold by 10 mg/kg 5-MTHF and by 1.4-fold by donepezil treatment . Asp levels increased by 21.7% in AD rats relative to controls (p < 0.001) but were decreased by treatment with 5 mg/kg 5-MTHF to 82.3% and with donepezil to 86.1% (p < 0.05). Glu was 1.3-fold higher, Gly 1.4-fold higher and γ-GABA 1.7-fold higher in healthy rats than in the AD model rats (p < 0.05). Supplementation with 10 mg/kg 5-MTHF increased Glu by 28.0% (p = 0.002). HPLC separation chromatograms are shown in Differences in Asp, Glu, Gly and γ-GABA are shown in p < 0.01) but was increased by 10 mg/kg 5-MTHF and donepezil treatment (p < 0.05). Expression of BACE1 mRNA was higher in the hippocampus of AD model rats than in controls (p = 0.007) and was decreased by treatment with 5 and 10 mg/kg 5-MTHF (p < 0.05).As shown in 1-42 and p-Tau increases in a rat model of AD induced by D-gal and AlCl3 exposure, and the same treatment also ameliorated cholinergic damage and endothelial cell dysfunction, improved the numbers and structures of pyramidal cells in the hippocampal CA1 region and regulated oxidative stress and excitatory amino acid release.5-MTHF treatment was found to decrease memory dysfunction and restrict Aβ3 and delayed AD development.Morris water maze testing illustrated the increased latency of AD rats, consistent with previous research , indicat1-42 and p-Tau have been previously shown to increase in AD rats [1-42 may result from the decreased APP promoter methylation caused by AlCl3, which increases APP mRNA and promotes Aβ deposition [1-42 and p-Tau were decreased by MTHF supplementation, and 5-MTHF may slow Aβ accumulation and p-Tau production by reducing APP levels and glycogen synthase kinase-3β activation, alleviating memory impairment.Pathological changes involved in AD typically include senile plaques composed of accumulated Aβ deposits and neurofibrillary tangles containing p-Tau . Aβ1-42  AD rats . Increasposition . In addiposition . Aβ1-42 κB (NF-κB) [κB pathway.Increased AChE activity was observed in the current cohort of AD rats, in agreement with previous findings , and the (NF-κB)  to enhanOxidative stress has been considered a risk factor for aging and various neurodegenerative diseases, including AD . It may κB activation [Endothelial cells are an essential part of the cardiovascular system, and they synthesize and release a variety of biologically active substances. However, abnormal cytokine secretion may lead to endothelial dysfunction, and this condition has been linked to AD . Endothetivation  and aggrtivation . 5-MTHF tivation .The hippocampus of the brain’s limbic system is involved in learning, memory and spatial orientation , and theBrain amino acid neurotransmitters contribute to neuronal information transmission and cognitive activity . ExcitatAPP metabolism consists of the production of soluble products by the α-secretase ADAM10 and the production of Aβ by the β-secretase BACE1 . HippocaA limitation of the current study concerns its multi-target approach to 5-MTHF, which covers a wide range of areas but does not include in-depth mechanistic analyses of AD pathogenesis. Further experiments are planned to scrutinize the mechanistic factors.1-42 and p-Tau in a rat model of AD induced by D-gal and AlCl3. SOD activity and VEGF levels were raised, and AChE and NOS activities and MDA, NO, ET-1, IL-6 and TNF-α levels were reduced by 5-MTHF treatment. The numbers and structures of pyramidal cells in the hippocampal CA1 region were restored and excitatory amino acid release and APP processing regulated.In conclusion, 5-MTHF alleviated memory impairment and restricted increases in Aβ


The optimized porous carbon material (COCLP-4.5), obtained by adjusting the raw material ratio of the organic crosslinked polymer, exhibits a high specific capacitance (522 F·g−1 at 0.5 A·g−1), good rate capability (319 F·g−1 at 20 A·g−1), and outstanding stability (83% retention after 5000 cycles) in a three-electrode system. Furthermore, an energy density of 18.04 Wh·kg−1 is obtained at a power density of 200.0 W·kg−1 in a two-electrode system. This study demonstrates that organic crosslinked polymer-derived porous carbon electrode materials have good energy storage potential.Supercapacitors, as a new type of green electrical energy storage device, are a potential solution to environmental problems created by economic development and the excessive use of fossil energy resources. In this work, nitrogen/oxygen (N/O)-doped porous carbon materials for high-performance supercapacitors are fabricated by calcining and activating an organic crosslinked polymer prepared using polyethylene glycol, hydroxypropyl methylcellulose, and 4,4-diphenylmethane diisocyanate. The porous carbon exhibits a large specific surface area (1589 m Solutions to environmental problems, owing to economic development and the excessive use of fossil energy resources, are urgently being sought . SupercaTheoretical research on and practical applications of supercapacitors have significantly progressed; however, insufficient energy density and high cost are still challenges requiring resolution ,6,7. Ele−1 at 0.5 A·g−1 in a three-electrode system. The symmetrical supercapacitor device assembled with this nitrogen-doped carbon also displayed good performance, with an energy density of 16.3 Wh·kg−1 at a power density of 348.3 W·kg−1.Nitrogen doping has been demonstrated to be an effective way to improve the wettability and conductivity of carbon materials and can also provide additional pseudocapacitance for supercapacitors. Generally, nitrogen-doped carbon materials can be prepared using two synthetic strategies, namely by the pyrolysis of nitrogen-containing precursors, such as biomass , synthet−1 at a current density of 0.5 A·g−1 in a three-electrode system. Chen et al. [−1 at a current density of 0.5 A·g−1.Organic crosslinked polymers are mainly composed of elements, such as carbon, nitrogen, oxygen, and hydrogen, which have the characteristics of a network structure. Porous carbon materials prepared using such polymers had a high heteroatom content, specific surface area, and outstanding electrochemical properties . In partn et al.  prepared2·g−1) and a high specific capacitance of 522 F·g−1 at a current density of 0.5 A·g−1. Furthermore, its energy density reached 18.04 Wh·kg−1 at a power density of 200.0 W·kg−1 in a two-electrode system using 1 M Na2SO4 as the electrolyte. Mechanistic studies showed that the high electrochemical performance of the obtained carbon was attributed to the network structure and rich N/O content of the crosslinked polymer. Hence, the preparation method for porous carbon materials proposed in this study provides a new approach for the research and development of electrode materials.In this work, nitrogen/oxygen(N/O)-doped carbon-based porous materials were fabricated by carbonizing and activating an organic crosslinked polymer with a network structure. The organic crosslinked polymer was synthesized using polyethylene glycol (PEG 6000), hydroxypropyl methylcellulose (HPMC), and 4,4-diphenylmethane diisocyanate (MDI). The carbon material obtained by optimizing the ratio of the raw materials had a large specific surface area  were purchased from Aladdin. Analytical-grade potassium hydroxide (KOH) and acetylene black were obtained from Xilong Science Co., Ltd. . None of the purchased reagents were purified before use. All aqueous solutions were prepared using ultrapure water .Polyethylene glycol , 4,4-diphenylmethane diisocyanate , hydroxypropyl methylcellulose , polytetrafluoroethylene (PTFE), and 3.5, OCLP4.5 and OCLP5.0, respectively. The organic crosslinked polymers were prepared by a one-pot method, which is a minor modification based on our previous report . Briefly2 atmosphere at a heating rate of 5 °C/min. The resulting carbon precursors were homogeneously ground with KOH in a mass ratio of 1.0:3.0, then calcined in a tube furnace at 600 °C under a N2 atmosphere for 2 h. The calcined products were stirred with a 1 M hydrochloric acid solution for 2 h, followed by washing with distilled water and anhydrous ethanol sequentially until the filtrate was neutral. The obtained residues were dried in a blast oven at 80 °C for 24 h to obtain porous carbon materials, which were named as COCLP-3.5, COCLP-4.5, and COCLP-5.0, respectively.The prepared OCLPs were directly carbonized by heating them in a tube furnace at 500 °C for 2 h under a NFourier transform infrared (FTIR) spectroscopy was performed on the samples using a Thermo Fisher  Nicolet 6700 spectrometer with KBr pellets. A powder X-ray diffractometer  operating at 40 kV and 40 mA with Cu Kα radiation (λ = 0.15406 nm) in the 2θ range of 5–90° with 0.01° step increments was used to analyze the microstructure of the materials. The chemical structure and graphitization of the samples were further characterized using Raman spectroscopy  at an excitation wavelength of 532 nm. The surface micromorphology of the samples was characterized using scanning electron microscopy  and transmission electron microscopy , and elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDS). The specific surface area and pore structure characteristics of the samples were characterized using a nitrogen adsorption–desorption analyzer  at −196 °C. Surface element analysis was performed using X-ray photoelectron spectroscopy .OCLP), acetylene black, and PTFE in a weight ratio of 8:1:1 was applied to nickel foam (2 cm × 2 cm) as the working electrode; platinum and Hg/HgO electrodes were used as the counter and reference electrodes, respectively, in the three-electrode system. The voltage was set to −1–0 V and the electrolyte was 6 M KOH. A symmetric supercapacitor was built for a two-electrode system using the COCLP, a 1 M Na2SO4 electrolyte, and a voltage range of 0–1.6 V.The electrochemical performance of the samples, including galvanostatic charge–discharge (GCD), cyclic voltammetry (CV), and electrochemical impedance (EIS), was measured using a CHI 660E instrument in a three-electrode system. A slurry mixture of carbon material (C−1) of the electrode material were calculated based on the GCD curves using Equations (1) and (2), respectively.I (A), Δt (s), ΔV (mV), and m (g) represent the discharge current, discharge time, discharge voltage range, and mass of the active material of a single electrode, respectively.For the three-electrode and two-electrode systems, the weight-specific capacitances (F·gcellE) and power density (cellP) of the symmetrical supercapacitor were calculated using Equations (3) and (4), respectively.Cg is obtained from Equation (2), ΔV is the working voltage of the discharge, and Δt is the discharge time.The energy density  are similar. The peaks around 3438, 1639, 1526, and 1106 cm−1 correspond to the stretching vibration absorption peaks of the –OH, C=O, C–N, and C–O groups, respectively, which is consistent with the organic crosslinked polymer [ polymer . The aboOCLPs obtained from the OCLPs were characterized using XRD and Raman spectroscopy. OCLP-3.5, COCLP-4.5, and COCLP-5.0, showing that all the COCLPs exhibit obvious diffraction peaks at 43°, corresponding to the (100) crystal planes of the graphite structure. The results indicate that COCLP-3.5, COCLP-4.5, and COCLP-5.0 have amorphous graphite structures [OCLP-4.5 is the weakest, demonstrating that COCLP-4.5 has the highest structural disorder [−1, corresponding to the D and G peaks of graphite, respectively. The ratio of the areas of the D peak to the G peak (D/AGA) reflects the order degree of the COCLP structure [OCLP-3.5, COCLP-4.5, and COCLP-5.0 are 1.15: 1, 1.18: 1, and 1.13: 1, respectively. This result also illustrates that COCLP-4.5 has more defects because the D peak represents a defect peak caused by the low symmetry or irregularity of the carbon material [The Cructures ,29. The disorder . Figure tructure . The calmaterial .OCLP-3.5, COCLP-4.5, and COCLP-5.0 were characterized using SEM, as shown in OCLPs are all porous and present a three-dimensional network structure. The number of pores in the COCLP increases with an increase in the amount of HPMC; however, when the HPMC content is increased to 5.0 g, the pore structure is only partially formed, and the number of pores decreases. The result demonstrates that the pore structure of COCLP-4.5 was excellent. Generally, an abundant number of pores can significantly increase the specific surface area of COCLPs, thereby providing more storage sites and transport channels for electrolyte ions. This is beneficial for improving the electrochemical performance [The surface morphologies of Cformance .OCLP-4.5 is a porous COCLP. When COCLP-4.5 is used as the electrode material, these disordered microporous structures can provide sufficient active sites for charge storage [OCLP-4.5, showing that carbon, nitrogen, and oxygen were uniformly distributed in the carbon framework. Abundant nitrogen and oxygen can introduce pseudocapacitance and enhance the capacitance performance of the electrode material.Additionally,  storage . Figure OCLPs were subjected to N2 adsorption–desorption measurements to explore the pore characteristics. OCLPs exhibit obvious type I isotherm characteristics, indicating that these samples are rich in micropores [OCLPs, showing that the specific surface area and pore volume of these samples are mainly provided by the micropores and mesopores. Among the three samples, COCLP-4.5 has the largest specific surface area (1589 m2·g−1) and the highest pore volume (0.657 cm3·g−1), which further confirm that COCLP-4.5 has the best pore structure. Numerous studies have demonstrated that the large specific surface area and rich pore structure of porous carbon material can greatly promote the storage and rapid migration of ions, resulting in the excellent specific capacitance performance of supercapacitors [2SO4, acidic), KOH , and sodium sulfate . The electrolyte ions in these electrolytes mainly exist as hydrated ions  at a sweep rate of 5 mV·s−1. All the samples display a typical rectangular shape, indicating that the capacitive behavior of these materials is mainly electric double-layer capacitance. Concurrently, these curves have a broad peak in the voltage window of −0.8 to −0.3 V, which is caused by the oxidation–reduction reaction of nitrogen and oxygen atoms contained in these samples during the charge and discharge process. Moreover, the pseudo-capacitance introduced by the redox reaction can significantly increase the specific capacitance of carbon electrodes. The COCLP-4.5 sample exhibits the largest encircled area of the CV curve among the three samples, which also illustrates that COCLP-4.5 has the highest specific capacitance. OCLPs at a current density of 1 A·g−1. The GCD curves for the three samples are all quasi-isosceles triangle shapes, indicating that the capacitance is mainly electric double-layer capacitance (EDLC), and the slight deformation is attributed to the existence of pseudo-capacitance. According to Equation (1), the specific capacitances of COCLP-3.5, COCLP-4.5, and COCLP-5.0 at a current density of 1 A·g−1 are 302, 503, and 330 F·g−1, respectively. This result shows that the specific capacitance of COCLP-4.5 is the largest, owing to its large specific surface area (1589 m2·g−1) and pore volume (0.657 cm3·g−1). OCLP-4.5 at different scanning rates. It reveals that the COCLP-4.5 still maintains a quasi-rectangular shape at scan rates of 5–50 mV·s−1, indicating that the good pore structure of COCLP-4.5 enables the rapid migration of electrolyte ions to result in its good rate capability. OCLP-4.5 at current densities of 0.5–20 A g−1, showing that the GCD curve does not exhibit a significant IR drop at a high current density of 20 A·g−1. Therefore, it demonstrates that the COCLP-4.5 has a high conductivity, good rate capability, and electrochemical reversibility. The specific capacitances are calculated as 522, 503, 432, 396, 363, and 319 F·g−1 at current densities of 0.5, 1, 2, 5, 10, and 20 A·g−1, respectively. Comparing the electrochemical performance of COCLP-4.5 with that of the references, the result is listed in OCLP-4.5 is better than that of other electroactive materials reported in the literature. This is attributed to the unique network structure and rich N/O content of the crosslinked polymer fabricated in this study.The electrochemical performances of the electrode materials were evaluated using a three-electrode system. OCLPs and the equivalent circuit model  and Rs (contact resistance with the electrolyte) among the three materials. That is, in the high-frequency region, the Rct of COCLP-4.5 is 0.042 Ω, lower than those of COCLP-3.5 (0.152 Ω) and COCLP-5.0 (0.183 Ω). The low Rs demonstrates that the electrolyte ions are readily transferred to the surface of the COCLP-4.5 electrode [OCLP-4.5 is almost vertical in the low-frequency region. The EIS results illustrate that the structure of COCLP-4.5 is beneficial for charge transfer and the efficient diffusion of electrolyte ions. For supercapacitors, the cycling stability is a significant parameter to estimate their practical application. OCLP-4.5 retains 83% of its initial specific capacitance value after 5000 cycles at a current density of 5 A g−1. The surface morphology of COCLP-4.5 after cycling was characterized by SEM, as shown in OCLP-4.5, before , the specific capacitance of COCLP-4.5 is 203 F·g−1 at 1 A·g−1 and its specific capacitance remains 150 F·g−1 at 10 A·g−1, demonstrating a good rate capability even at high current densities for the symmetric supercapacitor. −1. It displays that the capacitance retention of the device is 84.0% after 5000 cycles, reflecting good cycling stability. −1 at a power density of 200.0 W·kg−1 based on Equations (3) and (4), significantly higher than those reported in recent years . SpecifOCLPs. The results indicated that the COCLP-4.5 obtained by optimizing the raw materials exhibited an excellent electrochemical performance. For instance, the specific capacitance of COCLP-4.5 was as high as 522 F·g−1 at a current density of 0.5 A·g−1, and still exhibited 309 F·g−1 at 20 A·g−1 in a three-electrode system. Furthermore, the symmetric capacitor achieved an energy density of 18.04 Wh·kg−1 at a power density of 200.0 W·kg−1. The COCLPs benefitted from the net structure of organic crosslinked polymers to form hierarchical porous carbon and the pseudocapacitance introduced by heteroatoms. Therefore, the method for fabricating carbon material proposed in this study provides a new strategy for the development of electrode materials with high electrochemical performance.In this study, a network-structured organic crosslinked polymer was used as a carbon source to obtain N/O-doped porous C