N-glycosyltransferases (NGTs) catalyze the glycosylation of Asn residues in proteins, a process fundamental for bacterial autoaggregation, adhesion and pathogenicity. However, our understanding of their molecular mechanisms is hindered by the lack of structures of enzymatic complexes. Here, we report structures of binary and ternary NGT complexes of Aggregatibacter aphrophilus NGT (AaNGT), revealing an essential dyad of basic/acidic residues located in the N-terminal all α-domain (AAD) that intimately recognizes the Thr residue within the conserved motif Asn0-X+1-Ser/Thr+2. Poor substrates and inhibitors such as UDP-galactose and UDP-glucose mimetics adopt non-productive conformations, decreasing or impeding catalysis. QM/MM simulations rationalize these results, showing that AaNGT follows a SN2 reaction mechanism in which the acceptor asparagine uses its imidic form for catalysis and the UDP-glucose phosphate group acts as a general base. These findings provide key insights into the mechanism of NGTs and will facilitate the design of structure-based inhibitors to treat diseases caused by non-typeable H. influenzae or other Gram-negative bacteria.Soluble HMW1C-like  NGTs glycosylate asparagine residues in proteins, crucial for bacterial adhesion and pathogenicity. Here, the authors provide insights via crystallography and simulations, showing acceptor asparagine uses imidic form for catalysis and UDP-glucose phosphate group acts as general base. N-glycosylation, is a widely occurring post-translational modification found in most eukaryotes1 and some prokaryotes3. It has been reported that more than 7000 human proteins are N-glycosylated4. These N-glycans play essential roles in cellular function, mostly in eukaryotes, and are involved in processes such as protein folding and stability, protein trafficking, and signal transduction, playing a major role in health and disease6. Together with protein O-glycosylation, N-glycosylation is present in most approved or preclinical protein therapeutics10. It affects immunogenicity11 and potency12, motivating the close study of glycosylation pathways and glycosylation mechanisms13.The attachment of sugar molecules to asparagine residues in proteins, also known as N-glycosylation is initiated by the membrane-bound oligosacharyltransferase enzyme (OST), which attaches a preassembled oligosaccharide to Asn residues in nascent glycoproteins. OST is a complex oligomeric enzyme in proteins of animals, plants, and fungi14, whereas it is a monomeric enzyme in bacteria, archaea, and protozoa16. It was discovered in 200317 that bacteria are able to perform a simpler version of N-glycosylation in which a single monosaccharide or a disaccharide, rather than a complex oligosaccharide, is attached to specific asparagine residues. This process is catalyzed by soluble N-glycosyltransferase enzymes (NGTs). Bacterial N-glycosylation, which is either accomplished by OSTs or NGTs, is important for bacterial survival, adhesion, autoaggregation, and pathogenicity18.Haemophilus influenzae17. This enzyme, termed HMW1C, was demonstrated to be capable of adding mono- and di-hexose units onto Asn residues of proteins, such as HMW1 adhesin18. Similar enzymes were later discovered in many other Gram-negative bacteria, including Actinobacillus pleuropneumoniae19, Haemophilus ducreyi and Mannheimia haemolytica20, Yersinia pestis and Escherichia coli10, Aggregatibacter aphrophilus11, Kingella kingae, and Bibersteinia trehalosi21.The first known example of an NGT was discovered in non-typeable 23. They also share the Asn0-X+1(X ≠ P)-Ser/Thr+2 conserved sequence motif in their protein acceptor substrates24  and a C-terminal catalytic domain that adopts a GT-B fold, as shown for Actinobacillus pleuropneumoniae NGT (ApNGT)23. In addition, while the OSTs require a metal, preferably Mn+2, to catalyze the reaction, no metal is necessary for the NGTs18.Although OSTs and NGTs differ in the type of donor substrate (lipid-bound sugars for OSTs versus nucleotide-bound sugars for NGTs), they are both inverting GTs, i.e. they catalyze the formation of a glycosidic bond with inversion of configuration of the donor anomeric carbons24 Fig. . The numApNGT and in complex with UDP, the peptide substrate recognition and the catalytic mechanism of either OSTs or NGTs enzymes remain unknown27. Most inverting GTs follow a mechanism in which a catalytic base deprotonates the incoming nucleophile of the acceptor , an inverting GT that is an orthologue of ApNGT. Our results indicate that Thr residues at positions +2 and +3 are the main determinants of peptide-NGT interactions, while poor donor substrates such as UDP-galactose , and inhibitors such as UDP-Glc mimetics, adopt unproductive conformations that compete with the peptide substrate, resulting in reduced or impeded catalysis, respectively. QM/MM metadynamics simulations show that AaNGT follows a concerted single-displacement SN2 mechanism in which the acceptor Asn attacks the sugar donor anomeric carbon via the imidic form and the α-phosphate of UDP-Glc acts as the catalytic base.Here, we have employed a multidisciplinary approach involving structural biology, synthetic chemical biology, kinetic experiments, and computational techniques to uncover the recognition of substrates and the catalytic mechanism of AaNGT, we designed a full-length construct that was expressed in E. coli , being the latest prepared according to our previous methodologies29  experiments with the sugar nucleotides, UDP and the peptide. While reasonable titration curves were obtained with the sugar nucleotides and UDP , the protein O-fucosyltransferase SPINDLY from Arabidopsis thaliana (PDB entry 7Y4I) and the Xanthomonas campestris putative O-GlcNAc transferase, OGT . As expected, ApNGT has the lowest RMSD of 1.4 Å on 618 aligned residues, due to the high structural homology between both NGTs . This is likely due to their different GT-B fold catalytic domains, as these GTs bind to different sugar nucleotides and glycosylate different protein substrates34. Furthermore, NGTs possess a distinct N-terminal AAD domain, while XcOGT and SPINDLY contain N-terminal TPRs, which differ in fold from the AAD domain.Having characterized AaNGT  exhibits more interactions than in the binary complexes, with Thr519, Asn520, and Gly521 backbones, and Ser277, Thr437, Lys440, and Asn520 side chains contributing to this. In contrast, the UDP-Gal and UDP-2F-Glc pyrophosphates are only recognized by the side chains of Lys440 and Asn520. The sugar molecules interact with the side chains of His276 and Ser277. Furthermore, the Gal moiety specifically interacts with the Gly369 backbone, while the 2F-Glc moiety interacts with the side chain of His370 . These interactions include the backbone atoms of Phe1-2 and Ser277, the backbone of Asn30 and the UDP α-phosphate, the side chain of Asn30 and Gly369, the backbone of Thr5+2 and the Arg177 side chain, the Thr5+2 and Asp215 side chains, the Thr6+3 backbone and the Arg177 side chain, and the Thr6+3 and His214 side chains . The latter residue is located at the peptide binding site and its importance for enzyme activity was previously demonstrated. In fact, the equivalent residue in ApNGT (Gln469) was found to be deleterious for NGT activity, whereas its mutation to Ala significantly improved NGT activity35. The superposition of all ligands for all three structures  clearly shows that the sugar moieties of UDP-Gal and UDP-2F-Glc collide with Gly2-1 and Asn30  is located in front of the UDP pyrophosphate, although its side chain does not participate in interactions with any residue of AaNGT that could potentially serve as a catalytic base. Consequently, our ternary complex does not supply any evidence on how the Asn is glycosylated and thus no insights into the NGT catalytic mechanism could be inferred.In summary, the structural analysis shows that NGT shares two critical and conserved residues among NGTs  due to strong steric clash between the sugar and the peptide . O-GlcNAc transferase is an inverting glycosyltransferase with a GT-B fold highly similar to that of AaNGT. It is noteworthy that despite OGT catalyzing a distinct reaction (O-glycosylation), both enzymes are classified in the same CAZy family, GT41, owing to their similarities at the catalytic domain level36. MD simulations for up to 400 ns  is located on the β face of the donor sugar, opposite to the sugar-phosphate bond, with the amino group close to the Glc anomeric carbon  and the recently investigated plant protein O-fucosyltransferase SPINDLY 37. The possible catalytic base character of the α-phosphate is further supported by NMR and computational experiments that demonstrate that the pKa of the α-phosphate in UDP, UDP-GlcNAc, and UDP-S-GlcNAc is ∼6.539.As inferred from the crystal structure complexes, no residue serving as a general catalytic base in the anticipated SAaNGT, we selected one representative snap-shot of the classical MD simulation and performed quantum mechanics/molecular mechanics (QM/MM) MD simulations (for 5 ps), using a QM region that included most of the donor molecule (Glc and the two phosphate groups), and the side chain of Asn30 . The active site remained in a similar configuration as in the previous MD simulations, in which the Asn30 amino group is engaged in a hydrogen bond with the α-phosphate. Afterwards, we tried to model the N-glycosylation reaction using QM/MM metadynamics, as well as other enhanced-sampling methods (see Methods) previously used to study glycosylation reactions43. Unfortunately, all our attempts to obtain a feasible reaction pathway failed. Even though the Asn30 residue became glycosylated during the simulation .To model the reaction mechanism of N-glycosylation reaction resulted in tautomerization of the amide group of Asn30 via one oxygen atom of the α-phosphate. In other words, one of the amide protons transferred to Oα and, subsequently, the amide carbonyl abstracted the proton from Oα, resulting in the imidic form of Asn30. It was interesting to observe that the N atom of the imidic Asn is better poised for nucleophillic attack than the Michaelis complex with Asn30 in the amide form. In particular, the NAsn-C1-O1 angle in the imidic Asn increases by ≈ 15° (from 115° to 130°) with respect to the angle in the amide complex . We used two collective variables corresponding to the main covalent bonds that need to be formed or broken during the reaction: the nucleophilic attack distance (N-C1) and the leaving group distance (C1-OP)  to the reaction products (AaNGT + UDP + Glc-peptide), in which Asn30 is glycosylated  is still somewhat higher than the one estimated from the experimental rate constant 45, probably due to an imperfect position of the phosphate groups in the initial structures. However, it is similar to the one previously computed for OGT 46. Most importantly, the free energy barrier is much reduced compared to the one obtained for the reaction via the amide form of Asn30 , indicating that the reaction occurs preferably via the imidic form of Asn30.To evaluate the possibility of OP) Fig. . During ted Fig. . The reated Fig.  shows a +1 is present at the MC, which probably contributes to position the peptide in the active site. This might explain recent experimental results that show that particular peptide sequences with a Trp at that position improves the glucosylation efficiency20. The computed reaction pathway also shows a TS in which the sugar-phosphate bond is being broken (C1-OP = 2.2 Å) and the sugar-Asn30 bond is being formed (C1-N = 2.1 Å) .Representative structures of the MC, transition state (TS), and reaction products (P) are shown in in Fig. AaNGT active site. To this end, we performed QM/MM metadynamics simulations of the tautomerization process considering two possible scenarios: tautomerization mediated by the α-phosphate or tautomerization via active site water molecules  of proteins47. However, it is intriguing to understand why Nature has chosen the amide group of Asn residues, which is one of the least reactive nucleophiles in proteins, for glycosylation. Although the exact reason for selecting Asn residues for glycosylation remains unknown, we can investigate the mechanistic strategies utilized by various types of GTs to accomplish this PTM, which have arisen throughout evolution and are widespread in eukaryotes and certain bacteria. These GTs include single or complex membrane-bound oligosaccharyltransferases (OSTs) and soluble N-glycosyltransferases (NGTs).N-glycosylation catalyzed by NGTs. We focused on AaNGT, an inverting GT that catalyzes the transfer of Glc from UDP-Glc to Asn residues in the consensus sequence Asn-X-Ser/Thr. We provide high-resolution crystal structures for the enzyme in complex with a poor donor substrate  and a donor mimic (UDP-2F-Glc), as well as the enzyme in complex with a peptide acceptor (FGNWTT) and UDP, together with kinetic/ITC experiments and QM/MM metadynamics simulations of the reaction mechanism. Our results indicate that Thr residues at positions +2 and +3 are the main determinants of peptide-NGT interactions and the AAD domain recognizes these residues. Donor substrates less favorable than UDP-Glc, like UDP-Gal and inhibitors such as UDP-Glc mimetics, adopt unproductive conformations that compete with the peptide substrate, ultimately leading to decreased or hindered catalysis. These results are confirmed by MD simulations of the reconstructed Michalis complex 49, cellulase Cel45A50 and GH85 endo-β-glucosaminidases51.QM/MM metadynamics simulations of the reaction mechanism showed that the reaction starting from the most common amide of Asn15, but is similar to the one proposed37 (and confirmed by QM/MM simulations46) for O-glycosylation catalyzed by OGT. Additionally, we found that NGTs recognize Thr+2 differently from OSTs26, suggesting that the two enzymes might employ distinct mechanisms to achieve N-glycosylation.Another significant discovery from our study is the identification of the α-phosphate as the base responsible for deprotonating the acceptor Asn. This differs from the proposed mechanism for OSTs, which involves a twisted amide promoted by the binding of the amide group to metal-coordinated acidic residuesN-glycans in combination with endo-β-N-acetylglucosaminidases (ENGases) and synthetic oligosaccharides. This approach has proven useful in enhancing the stability of therapeutic peptides such as glucagon-like peptide 152. Moreover, NGTs have been employed alongside subsequent GTs to produce custom-made glycoproteins and glycoprotein-based nanomaterials with potential biomedical applications53. However, it is worth noting that NGTs primarily add glucose onto Asn residues, while GlcNAc residues are more desirable in certain cases for N-glycans in proteins from eukaryotes. Our structural and mechanistic insights might provide a potential solution to facilitate the engineering of NGTs to achieve N-GlcNAcylation. Furthermore, our work also opens up possibilities for synthetizing structure-based inhibitors to treat diseases caused by non-typeable H. influenzae or other Gram-negative bacteria, considering the high similarity between the active sites of NGTs.NGTs are not only interesting for their intriguing structural and mechanistic aspects, but also for their biotechnological properties as possible therapeutic targets for treating infectious diseases. In terms of biotechnological applications, NGTs have been targeted to synthesize more homogeneous AaNGT and propose that the enzyme follows a concerted single-displacement mechanism using one UDP phosphate group as general base. Additionally, we highlight the importance of the tautomeric form of Asn acceptor residues as a necessary step for glycosylation. This study exemplifies how GTs employ different strategies to activate less reactive nucleophilic groups such as Asn residues to achieve glycosylation.In summary, our experimental and computational work reveals the molecular basis of UDP-Glc and peptide recognition by AaNGT protein (aa 1-621) was codon optimized and synthesized by GenScript (USA) for expression in E. coli cells. This construct was a gift from Dr. Min Chen at Shandong University30. The construct was subcloned in pMALC2x, rendering the vector pMALC2x-10Hist-PP-AaNGT. The plasmid contained a sequence encoding a 10xHis tag and a PreScission protease (PP) cleavage site between the maltose binding protein (MBP) and the protein of interest. AaNGT mutants  were generated by GenScript via site-directed mutagenesis using the vector pMALC2x-10HistTAG-PP-AaNGT.The DNA sequence encoding amino acid residues of the d-thiogalactoside) and incubated at 18 °C for 18 h. The cells were harvested by centrifugation at 17,700 × g at 4 °C for 10 min and resuspended in buffer A . The protein was then loaded onto a His-Trap column  and eluted with an imidazole gradient from 10 mM to 500 mM . Buffer exchange to buffer C  was carried out using a HiPrep 26/10 Desalting Column . To remove the MBP, the protein PP was added to the fusion protein and the mix was incubated at 4 °C for 18 h. The cleavage of the fused protein was confirmed with a SDS-page gel and the MBP was removed with a His-Trap Column. The fractions containing the AaNGT protein were then concentrated to ~2.5 mL using centrifugal filter units of 30,000 MWCO cutoff (Millipore). Subsequently, gel filtration chromatography was carried out using Superdex 75 XK26/60 column (Cytiva) in buffer C to further remove impurities. The protein was then concentrated once again and protein concentration was measured by absorbance at 280 nm and by using its theoretical extinction coefficient (ε280nmAaNGT = 67270 M−1cm−1). Note that a similar extinction coefficient was used to determine the concentration for the mutants. If the protein was used to produce crystals, it was buffer exchanged to buffer D (25 mM TRIS pH 7.5) prior to get concentrated. Additionally, the purity of the proteins was confirmed by SDS-PAGE.The plasmids were transformed into BL21 (DE3) Gold cells and colonies were selected on LB/Agar plates containing 100 μg/ml of ampicillin. The cultures were grown at 37 °C in 2XTY medium  containing 100 μg/ml of ampicillin. When the optical density of the cultures reached 0.6, they were induced with 1 mM IPTG  at 25 °C with AaNGT at 100 μM and UDP at 1.2 mM, UDP-2F-Glc/UDP-5S-Glc at 4 mM and UDP-Gal at 3.5 mM in 25 mM TRIS pH 7.5 150 mM NaCl. All the experiments were repeated at least two times independently with similar results, and one representative plot with the derived dissociation constant KD and standard error of fitting for each experiment is shown , reactions contained 500 nM of the wild-type enzyme in 25 mM Tris pH 7.5, 150 mM NaCl, and 500 μM of the sugar nucleotides and the peptide FGNWTT. The reactions were incubated for 30 min at 37 °C and stopped using 5 μl of UDP-detection reagent at a 1:1 ratio in a white, opaque 384-well plate and incubated in the dark for 1 h at room temperature before measuring with a Synergy HT (Biotek). To estimate the amount of UDP produced in the glycosyltransferase reaction, we created a UDP standard curve. The values were corrected against the UDP-Glc (or other sugar nucleotides when applicable) hydrolysis and were fit to a non-linear Michaelis–Menten program in GraphPad Prism 8 software from which the Km, kcat, and Vmax along with their standard errors were obtained. All experiments were performed in duplicate.Enzyme kinetics for the wild type and mutants were determined using the UDP-Glo luminescence assays (Promega). Initially, the wild type and mutants were tested in reactions containing 500 nM of enzyme in 25 mM Tris pH 7.5, 150 mM NaCl, 500 μM of peptide FGN,N′-diisopropylcarbodiimide). The peptide was then released from the resin, and all acid-sensitive sidechain protecting groups were simultaneously removed using TFA 95%, TIS (triisopropylsilane) 2.5% and H2O 2.5%, followed by precipitation with cold diethyl ether. The crude products were purified by HPLC on a Phenomenex Luna C18(2) column  and a dual absorbance detector, with a flow rate of 10 mL/min.The peptide was synthesized by stepwise microwave-assisted solid-phase synthesis on a Liberty Blue synthesizer using the Fmoc strategy on Rink Amide MBHA resin (0.1 mmol). All other Fmoc amino acids (5.0 equiv.) were automatically coupled using oxyma pure/DIC (The peptide used in this work was dissolved at 100 mM in buffer 25 mM Tris-HCl pH 7.5. The pH of each solution was measured with pH strips and when needed adjusted to pH 7–8 through the addition of 0.1–5 μL of 2 M NaOH.AaNGT complexes were grown by sitting drop experiments at 18 °C by mixing 0.4 μl of protein solution (17 mg/mL AaNGT and 5 mM ligands in buffer D “25 mM TRIS pH 7.5”) with an equal volume of a reservoir solution. The crystals for the different complexes were obtained in different conditions. The crystals for the AaNGT-UDP-peptide complex were obtained in a 22% polyacrylic acid 5100 sodium salt, 100 mM HEPES sodium salt pH 7. We further soaked these crystals with the same condition saturated with peptide  and 25 mM UDP for 30 min before flash freezing in a cryoprotectant solution containing 20% ethylene glycol. The crystals for the AaNGT-UDP-Gal complex were obtained in 0.1 M magnesium chloride, 0.1 M Na HEPES pH 7.5, 10% (w/v) PEG 4000 solution. These crystals were further soaked 5 min with 37.5 mM UDP-Gal prepared in buffer D and flash frozen in the cryoprotectant solution. The crystals for the AaNGT-UDP-2F-Glc complex were obtained in a 0.1 M calcium acetate, 0.1 M sodium acetate pH 4.5, 10% (w/v) PEG 4000 solution. We further soaked these crystals with 30 mM UDP-2F-Glc for 5 min before flash freezing in the cryoprotectant solution.Crystals of the AaNGT were collected on synchrotron beamlines I03 of the Diamond Light Source  and XALOC beamline at the ALBA synchrotron  at a wavelength of 0.97 Å and a temperature of 100 K. XDS54 and CCP4 software packages55 were used for data processing and scaling. Relevant statistics are presented in Table 55 and PDB entry 3Q3E as a template was used to solve the crystal structures. Initial phases were further improved by cycles of manual model building in Coot56 and restrained refinement with REFMAC555. Further rounds of model building in Coot with TLS refinement in REFMAC5 were performed for all complexes. The crystal structures were validated with PROCHECK and model statistics are presented in Table AaNGT-UDP-Gal complex shows that 95.1%, 4.6%, 0.1%, and 0.2% of the amino acids are in most favored, allowed, generously allowed and disallowed regions, respectively. The Ramachandran plot for the AaNGT-UDP-2F-Glc complex shows that 90.6%, 8.8%, 0.4%, and 0.2% of the amino acids are in most favored, allowed, generously allowed and disallowed regions, respectively. The Ramachandran plot for the AaNGT-UDP-peptide complex shows that 90.1%, 8.6%, 1.0%, and 0.3% of the amino acids are in most favored, allowed, generously allowed and disallowed regions, respectively. The asymmetric unit of the P212121 crystals contained two molecules of AaNGT.Diffraction data for the three crystals of 1H and 19F NMR spectra were recorded at 400 and 376 MHz using a Bruker AVANCE 400 Plus Nanobay in chloroform-d or deuterium oxide. 13C and 31P NMR spectra were recorded at 101 and 162 MHz with the same instruments in chloroform-d or deuterium oxide. Chemical shifts are given in ppm (δ) and referenced to tetramethylsilane or to the internal solvent signal used as an internal standard. Assignments in the NMR spectra were made by first-order analysis of the spectra, and were supported by 1H−1H COSY, 1H−13C HMQC correlation results. High-resolution mass spectrometry was performed on a Waters Synapt G2-S HDMS spectrometer. Unless otherwise stated, all the commercially available solvents and reagents were purchased from FUJIFILM Wako Pure Chemical Corporation and Merck KGaA without further purification. During purification by silica gel column chromatography, the absorbances of all fractions were measured at 262 nm using a JASCO UV-2075 Plus detector.1-6 and 8-13; see Supplementary Figs. Synthesis of UDP-2F-Glc (7) and UDP-5S-Glc 14) and their precursor species  was dissolved in 1,4-dioxane (5.00 mL) and cooled at 0 °C. The solution was added (diethylamino)sulfur Trifluoride  and the mixture was then heated at 100 °C by irradiating microwave for 5 min. The mixture was diluted with dichloromethane and washed with ice water, aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 5:1 to 3:1 (v/v) hexane:ethyl acetate to give compound 2 ; 1H NMR  δ 5.77 , 5.37 , 5.06 , 4.45 , 4.29 , 4.14 , 3.86 , 2.18 , 2.09 , 2.08 , 2.04 ; 19F NMR  δ –200.9 .1,3,4,6-Tetra-2  was dissolved in DMF (900 μL) added ammonium carbonate . The mixture was then stirred at 20 °C for 8 h. The mixture was diluted with ethyl acetate and washed with water and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 6:1 to 3:2 (v/v) hexane:ethyl acetate to give compound 3 ; 1H NMR  δ 5.59 , 5.48 , 5.32 , 5.04 , 4.92 , 4.52 , 4.30–4.09 , 3.94 , 3.77 , 3.55 , 2.09 , 2.09 , 2.08 , 2.04 , 2.04 .Compound 3  and DMAP  were co-evaporated with dry toluene. The mixture was dissolved in dry dichloromethane (1.30 mL) and cooled at –10 °C. The solution was then added diphenyl chlorophosphate , and the mixture was stirred at 20 °C for 30 min. The mixture was diluted with dichloromethane and washed with ice water, aqueous 1 M HCl, aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 5:1 to 2:1 (v/v) hexane:ethyl acetate to give compound 4 ; 1H NMR  δ 7.40-7.22 , 6.14 , 5.56 , 5.07 , 4.62 , 4.15 , 4.00 , 3.79 , 2.01 , 2.05 , 2.03 ; 13C[1H] NMR  δ 170.5, 170.00, 169.5 (COCH3×3), 130.1, 130.0, 125.9, 125.9, 120.5, 120.4, 120.2, 120.2 (Ph), 94.7 , 86.6 , 70.0 , 69.6 (C-5), 67.0 , 60.9 (C-6), 20.8, 20.7, 20.6 (COCH3).Compound 4  was dissolved in ethyl acetate/methanol  and the flask was filled with argon. The solution was added Platinum oxide (IV) (15.2 mg) and the mixture was then stirred at 20 °C for 4 h in hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was added triethylamine  and stirred for 30 min. The solution was evaporated to give a syrup including compound 5 (Bis (triethylammonium) 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-α-d-glucopyranosyl phosphate).Compound 5  was co-evaporated with pyridine and dissolved in pyridine (500 μL). The solution was added to UMP-morpholidate , which evaporated with pyiridine, in the reaction flask. Moreover, 1H-tetrazole  was co-evaporated with pyridine and dissolved in pyridine (500 μL). The solution was transferred to the reaction flask by a syringe and the mixture was then stirred at room temperature for 39 h. The mixture was evaporated and the residue was purified by silica gel chromatography with 9:1 to 6:1 (v/v/v) acetonitrile:water. The absorbance of each fraction was measured at 262 nm and the combined fractions were evaporated. The residue was purified by silica gel chromatography with 15:2:1 to 6:2:1 (v/v/v) ethyl acetate:methanol:water to give compound 6 ; 1H NMR  δ 7.97 , 5.95–5.91 , 5.81 , 5.43 , 5.03 , 4.79–4.65 , 4.38–4.37, 4.34 , 4.25–4.22 , 4.17–4.09 ; 13C[1H] NMR  δ 171.4 (COCH3), 170.9 (COCH3), 170.6 (COCH3), 164.0 (C-4′′), 149.5 (C-2′′), 139.5 (C-6′′), 100.4 (C-1′), 89.9 , 86.4 (C-5′′), 84.5 , 80.8 , 71.8 (C-2′), 68.8 , 67.2 (C-3′), 65.8 (C-5), 65.4 , 62.7 , 59.3 (C-6), 18.0 (COCH3), 17.9 (COCH3), 17.8 (COCH3); 19F NMR  δ –200.9 ; 31P NMR  δ –11.59 , –13.89 .The mixture containing compound 6  was dissolved in triethylamine/methanol/water  and stirred at −20 °C for 12 h. The solution was then evaporated and lyophilized to give compound 7 ; 1H NMR  δ 7.88 , 5.94–5.90 , 5.74 , 4.46–4.29 , 4.31 , 4.23–4.11 , 3.97 , 3.86 , 3.80 , 3.72 , 3.47 , 3.14 3N), 1.21 3N); 13C[1H] NMR  δ 167.8 (C-4′′), 153.0 (C-2′′), 141.5 (C-6′′), 102.7 (C-5′′), 92.6 , 90.4–88.5 , 88.5 (C-1′), 83.1 , 73.7 (C-2′), 72.7 (C-5), 71.0 , 69.7 (C-3′), 68.6 , 65.0 , 60.1 (C-6); 19F NMR  δ –200.38 ; 31P NMR  δ –11.71 , –13.45 ; HRMS (ESI/Q-TOF) m/z: [M − H]− Calcd for C15H22F1N2O16P2−: 567.0434; Found: 567.0447.Compound d-glucopyranose 8  was dissolved in pyridine (1.80 mL) and added acetic anhydride . The mixture was stirred at 20 °C for 4 h. The mixture was then evaporated to give compound 9 ; 1H NMR  δ 6.15 , 5.89 , 5.44 , 5.39–5.27 , 5.24 , 5.11 , 4.38  4.31 , 4.15 , 4.06 , 3.59 , 3.31 , 2.08 , 2.07 , 2.04 , 2.04 , 2.02 , 2.02 , 2.01 , 1.99 .5-Thio-9  was dissolved in DMF (500 μL) added hydrazine monohydrate  and acetic acid . The mixture was then stirred at 20 °C for 30 min. The mixture was diluted with ethyl acetate and washed with water, aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 3:1 to 1:1 (v/v) hexane:ethyl acetate to give compound 10 ; 1H NMR δ  5.53 , 5.29 , 5.25–5.20 , 5.16–5.13 , 5.14 , 4.86 , 4.37 , 4.28 , 4.13–4.09 , 4.07 , 3.68 , 3.23 , 2.07-2.00 .Compound 10  and DMAP  were co-evaporated with dry toluene. The mixture was dissolved in dry dichloromethane (1.30 mL) and cooled at –10 °C. The solution was then added diphenyl chlorophosphate , and the mixture was stirred at 20 °C for 30 min. The mixture was diluted with dichloromethane and washed with ice water, aqueous 1 M HCl, aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 4:1 to 2:1 (v/v) hexane:ethyl acetate to give compound 11 ; 1H NMR  δ7.39–7.21 , 5.87 , 5.49 , 5.30 , 5.15 , 4.32 , 3.89 , 3.41 ; 13C[1H] NMR  δ 170.5, 169.8, 169.7, 169.5 (COCH3×4), 130.0, 125.9, 120.7, 120.7, 120.4, 120.4 (Ph), 78.0 , 73.8 , 71.4 (C-4), 70.20 (C-3), 60.69 (C-6), 39.73 (C-5), 20.7, 20.6, 20.6, 20.3 (COCH3×4).Compound 11  was dissolved in ethyl acetate/methanol  and the flask was filled with argon. The solution was added Platinum oxide (IV) (17.1 mg) and the mixture was then stirred at 20 °C for 4 h in hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was added triethylamine  and stirred for 20 min. The solution was evaporated to give a syrup including compound 12 (Bis (triethylammonium) 2,3,4,6-tetra-O-acetyl-5-thio-α-d-glucopyranosyl phosphate).Compound 12  was co-evaporated with pyridine and dissolved in pyridine (500 μ). The solution was added to UMP-morpholidate , which evaporated with pyiridine, in the reaction flask. Moreover, 1H-tetrazole  was co-evaporated with pyridine and dissolved in pyridine (500 μL). The solution was transferred to the reaction flask by a syringe and the mixture was then stirred at room temperature for 42 h. The mixture was evaporated and the residue was purified by silica gel chromatography with 9:1 to 6:1 (v/v/v) acetonitrile:water. The absorbance of each fraction was measured at 262 nm and the combined fractions were evaporated. The residue was purified by silica gel chromatography with 9:2:1 to 7:2:1 (v/v/v) ethyl acetate:methanol:water to give compound 13 ; 1H NMR δ  7.97 , 5.96–5.93 , 5.55 , 5.40 , 5.26 , 5.16 , 4.46 , 4.33–4.28 , 4.26–4.17 , 4.04 , 3.72 ; 13C[1H] NMR δ  173.7 (COCH3), 172.9 (COCH3), 172.8 (COCH3), 166.2 (C-4′′), 151.8 (C-2′′), 141.9 (C-6′′), 102.6 (C-5′′), 88.6 (C-1′), 83.0 , 74.6 , 74.3 , 73.9 (C-2′), 72.0 (C-4), 71.4 (C-3), 69.5 (C-3′), 65.0 , 61.3 (C-6), 38.3 (C-5), 20.3 (COCH3), 20.0 (COCH3), 20.0 (COCH3); 31P NMR  δ –11.59 , –13.40 .The mixture containing compound 13  was dissolved in triethylamine/methanol/water  and stirred at −20 °C for 12 h. The solution was then evaporated and lyophilized to give compound 14 ; 1H NMR  δ 8.04 , 5.94–5.91 , 5.54 , 4.41 , 4.35 , 4.30 , 4.21 , 4.00 , , 3.88–3.86 , 3.77 , 3.60 , 3.19–3.17 ; 13C[1H] NMR  δ 166.3 (C-4′′), 151.9 (C-2′′), 102.6 (C-5′′), 88.4 (C-1′), 84.0 , 82.4 (C-2), 78.3 , 74.4 (C-3), 73.9 (C-2′), 71.4 (C-4), 70.0 (C-3′), 63.3 , 59.5 (C-6), 43.9 (C-5); 31P NMR  δ13.21 , 2.89 (s); HRMS (ESI/Q-TOF) m/z: [M − H]− Calcd for C15H23N3O16P2S1−: 581.0249; Found: 581.0237.Compound 7) and UDP-5S-Glc (compound 14)  was not possible (the sugar moiety completely clashed with residues from the peptide). The structure was prepared for docking by removing subunit B, as well as the UDP molecules. The protonation of titratable residues  at pH 7 was decided by visual inspection as well as the online software MolProbity.The X-ray structure obtained in this work (PDB 8P0Q), containing the enzyme and the acceptor FGAaNGT+peptide was performed with AutoDock Vina57 and AutoDockTools 1.5.6. A group of selected residues near the active site were made flexible to better accommodate the substrate. Rotation of all the chemical bonds of UDP-Glc was allowed except for the dihedrals of the phosphates, to better mimic the phosphate configuration of UDP-GlcNAc of O-GlcNAc transferase (OGT)58. The values of the box size were taken as 32 x 30 x 26 Å, with the center of the box at 47.959, 62.228, 56.623 Å. Only one hit was obtained, with an affinity of –13.8 kcal/mol, which was used to perform molecular dynamics (MD) simulations.Docking of UDP-Glc into the structure of quitar subrallado 59. UDP charges were parametrized with Gaussian0960 and the antechamber program. The forcefields used for the protein, glucose, and UDP were F14SB61, GLYCAM0662, and GAFF63, respectively. TIP3P was used for water molecules64. The enzyme was solvated in a box of dimensions 97.868 × 112.646 × 114.217 Å, resulting in a total of 32129 water molecules. The system was neutralized by adding 18 sodium ions.The ternary complex obtained from the docking procedure was then prepared for classical MD simulations. The necessary files were obtained with the AmberTools suite59. The simulation procedure was as follows. First, the solvent was minimized without the protein and substrate, using positional restraints. Then, the whole system was minimized. Afterwards, the system was heated up to 300 K in a step-wise manner. The density of the system was taken to approximately 1 g/cm3. Subsequently, MD in the NPT ensemble was performed, restraining the distance between the donor and the acceptor for 60 ns before the restraint was released. The simulation was enlarged up to 460 ns, from which the last 400 ns were taken as production run. The evolution of the protein and acceptor peptide RMSD is provided in Supplementary Fig. Amber20 was used to perform the MD simulations0 in its tautomeric (imidic acid) form, following the same protocol as the one described above. Smooth restraints were applied in the distance between the donor and acceptor as well as the dihedral angles of the donor sugar were restrained for the first 50 ns of the MD simulation, followed by 150 ns of unrestrained MD, which was taken as production run. The system was found to be stable, with the Asn30 side chain well oriented for nucleophilic attack for most of the run. The evolution of the protein and acceptor peptide RMSD is provided in Supplementary Fig. Additional MD simulations were performed considering Asn365 interfaced with PLUMED 2.866. The QM/MM simulations combined Born-Oppenheimer molecular dynamics at DFT level for the QM atoms, handled with the QM program QUICKSTEP67, with classical MD for the rest of the system (simulated with the CP2K MM program FIST65). The QM region consisted of 44 atoms (counting capping atoms), including the donor glucose, two pyrophosphates, and the acceptor Asn (divided at the Cβ). The MM region included 106371 atoms. The boundary between the QM and MM regions was handled with the use of link H atoms via the Integrated Molecular Orbital Molecular Mechanics (IMOMM) method68. The QM region was enclosed inside a cell of 11.989 x 12.882 x 18.059 Å, and it was treated with the PBE functional69, along with DFTD3 corrections70, employing the GPW (Gaussian and plane-waves) scheme. GTH pseudopotentials71 were used, and the Gaussian triple-ζ valence polarized (TZV2P) basis set. A plane-wave cut-off of 400 Ry was employed, together with a MD time step of 0.5 fs. The simulations were performed in the NVT ensemble, using a Nosé-Hoover thermostat to keep the temperature around 300 K. Non-bonded cut-off was 12 Å.A representative snap-shot from the classical MD simulations in which the amide side chain interacts with the α-phosphate was selected to start the QM/MM MD simulations. All QM/MM simulations were performed using CP2K 9.172 simulations of the reaction the mechanism. The collective variable (CV) chosen was the difference between the Asn(N)-C1 and UDP-C1 distances. An upper wall was placed on distances related to the nucleophilic attack and deprotonation in order to limit the phase space accessible for the simulation . The Gaussian height was 1.0 kcal/mol, the width 0.1 Å and the deposition pace was set at 80 MD steps. The system reached the reaction products after having deposited 600 Gaussian functions (24 ps in terms of simulation time). However, the energy barrier was found to be very high , indicating that the reaction is not feasible. Thus, we stopped the simulation at this point. Alternative trials using different collective variables gave a similar negative result. Representative states of the reaction can be found in Supplementary Fig. The initial snapshot from the classical MD was optimized using the conjugate gradient algorithm, then equilbrated for 5 ps before starting the metadynamics73 is a recently developed technique, related to metadynamics, that in its “explore” formulation is very useful for a fast inspection of possible reaction mechanisms. An QM/MM OPES simulation of the reaction was performed starting from the same structure as the previous QM/MM metadynamics simulation. The same collective variable including the distance difference between the main bonds to be broken/formed (C1-OP and N-C1) was used. Additionally, since we knew from the previous simulation that the α-phosphate abstracts the amide proton, we used another collective variable that includes the two distances involved in the proton transfer (N-H and H–Oα). The previously described upper wall on distances was used. The OPES parameters used were 25 kcal/mol for the energy barrier, and 80 steps for the MD pace (40 fs). Surprisingly, the simulations show that Asn30 rapidly undergoes tautomerization before performing the nucleophilic attack. Even though the simulation was only exploratory, it gave us the idea that Asn could react in its imidic form.An alternative enhanced sampling method (OPES) was also used to model the glycosylation reaction, within the QM/MM formalism. OPESP) were used as collective variables. The same upper wall on distances was used. The metadynamics simulations were performed using a Gaussian height of 1.2 kcal/mol and a width of 0.1 (CV units) for both CVs, together with a pace of 80 MD steps (40 fs). A total of 2900 Gaussian functions were deposited, which in terms of real time amount to 116 ps. The simulation was stopped once the simulation crossed twice over the TS, as recommended for chemical reactions74. The location of the TS on the computed free energy landscape was further refined by committor analysis, using 30 independent replicas .An unbiased QM/MM MD (7.2 ps) of the system with imidic Asn was performed to check its stability, before another QM/MM metadynamics simulation of the chemical reaction was launched. Two distances involving in the nucleophilic attack, N-C1 and leaving group departure (C1-Op and N-H (CV1) and the difference in coordination numbers between OAsn-H and H–Op (CV2). The simulations used a Gaussian height of 0.8 kcal/mol, a width of 0.04 Å for both CVs, and a deposition pace of 100 MD steps (50 fs). The simulation run for a total of 166 ps, until recrossing over the transition state took place. The computed free energy barrier  indicated that tautomerization via the α-phosphate is not particularly favored (it is more difficult than tautomerization of amides in water solution75). Therefore, we launched a second simulation to assess whether tautomerization with the participation of water molecules was more likely. To this end, a larger QM region, including two active site water molecules (Wat1 and Wat2) and the Lys440 side chain (coordinating the pyrophosphate groups) was used. The QM/MM metadynamics simulation was performed using two CVs: CV1 = CN(N-H) + CN(HWat-OWat1) + CN(HWat2-OWat2) and CV2 = CN(H–OWat1) + CN(HWat1-OWat2) + CN(HWat2-O) (CN = coordination number). An upper wall was placed on distances related to the nucleophilic attack (not deprotonation) . The first CV is maximum when Asn is in its amide form, whereas the second one accounts for the imidic acid form. The simulation was performed using a Gaussian height of 1 kcal/mol and a width of 0.08 Å for both CVs, together with a deposition pace of 80 MD steps (40 fs). The simulation run for a total of 9.2 ps. In all cases, we used the following formula for coordination number . We used two collective variables; the difference in coordination numbers between H–Ond N-H CV and the Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


At the low sintering temperature, both  and  solid solutions were formed in the composites by adjusting the ratio of ZrC to TiC0.5N0.5. During the sintering process, the Si added at a rate of 5 mol% reacted with ZrC and TiC0.5N0.5 to generate SiC. With the increase in Si addition, it was found that the residual β-ZrSi was formed, which greatly reduced the flexural strength of composites but improved their toughness. The reaction and solid-solution-driven inter-diffusion processes enhanced mass transfer and promote densification. The solid solution strengthening and grain refinement improved the mechanical properties. The ZrC–47.5 mol% TiC0.5N0.5–5 mol% Si (raw powder) composite possessed excellent comprehensive performance. Its flexural strength, Vickers hardness, and fracture toughness were 508 ± 33 MPa, 24.5 ± 0.7 GPa, and 3.8 ± 0.1 MPa·m1/2, respectively. These reached or exceeded the performance of most  ceramics reported in previous studies. The lattice distortion, abundant grain boundaries, and fine-grained microstructure may make it possible for the material to be resistant to radiation.Dense and enhanced mechanical properties -based composites were fabricated using ZrC, TiC Zirconium carbide (ZrC) exhibits uniquely comprehensive properties such as a high melting point ~3540 °C), high hardness ~20 GPa), excellent abrasion and corrosion resistance, good thermal/electrical conductivities, and good irradiation damage tolerance ,2,3,4. C0 GPa, ex540 °C, h2 during sintering, which is conducive to the densification of ceramic.However, the densification temperature of ZrC is very high due to its strong covalent bond and low self-diffusion coefficient, which limits its application. Adding transition metal carbide, nitride, and carbonitride such as TiC, TiN, and TiCN to the ZrC matrix to form solid solutions can improve sintering performance ,12,13. TNevertheless, ZrC-TiCN ceramics have poor densification and mechanical properties due to the high sintering temperatures. Reactive hot-pressing is considered an effective method of preparing ceramic composites. Reactions during sintering can significantly enhance the low-temperature densification behavior compared with non-reactive processes ,20. For 0.5N0.5 should be 50:50 to obtain composites with Zr-rich  and Ti-rich  solid solutions that improve mechanical properties. ZrC-TiC0.5N0.5 (molar ratio 50:50) with 0, 5, 10, and 20 mol% Si additions were chosen as the designed compositions. The effects of Si addition on the microstructure and mechanical properties were investigated. This research will propose a new alternative for designing and preparing nuclear materials.In this study, dense -based composites were prepared by reactive hot-pressing at 1500–1700 °C. Si was chosen as a sintering aid to reduce the sintering temperature and enhance densification by using Si to generate the liquid phase during the sintering process. At the low sintering temperature, thermodynamic calculation showed that the molar ratio of ZrC to TiCdav = 0.25 μm, purity > 99.7 wt%), TiC0.5N0.5 , and Si  were chosen as the initial powders. The powder mixtures with different compositions were milled with a ball-to-powder ratio of 30:1 in weight under an argon atmosphere. The milling time was 24 h, and the rotating speed was 250 rpm. Then, the powders were passed through an 80-mesh sieve. Commercial ZrC  using a 20 mm inner diameter high-density graphite die. The room temperature was 20 °C, and the sintering curve of Z50T10S is shown in The entire surface of the samples was ground to remove the graphite foil residual and the reaction layer before being polished with diamond paste. An X-ray diffractometer  was used for identifying the phase constituents. The lattice parameters were determined using Si powder as the internal standard in the 2θ range of 20°–90°. The bulk densities of the specimens were measured by Archimedes’ principle. Microstructures were observed by a scanning electron microscope  equipped with energy-dispersive spectroscopy  for elemental analysis. The grain size and content of each phase in the specimen were obtained by image analysis using the Image-Pro Plus software (version 7.0). Transmission electron microscopy  was used for a more detailed analysis of the microstructure.3 test bars). The reported strength values were the average tested values on five bars where the surfaces were polished and chamfered. Vickers hardness (vH) was measured using the Vickers indentation method (HVS-30) with a load of 9.8 N for 10 s. Fracture toughness (ICK) was evaluated from the as-indented crack lengths by the Evans [vH and ICK were obtained from at least 10 indentations. The phase diagram based on thermodynamics was calculated by the software FactSage (Version 7.1) using databases from SpMCBN 7.0.The flexural strength was performed utilizing three-point bending (Instron-1186) with a 14 mm span and a crosshead speed of 0.5 mm/min  diffraction peaks mentioned above became progressively less shifted with the decreasing sintering temperature. The full width at half maximum of the  and  solid solutions (220) crystal plane diffraction peaks gradually decreases with the addition of Si. According to Scherrer’s formula, it can be assumed that the grain size of both solid solutions grows with the Si addition. systems ,29. The 0.5N0.5 lattice. Compared with the lattice parameters in the Z50T sample, those of the  and  rose from 0.4525 nm to 0.4589 nm and decreased from 0.4418 nm to 0.4346 nm in the Z50T20S, respectively. This is attributed to the solid solubility decreasing with the reduction in sintering temperature, which also made the offset of the corresponding (220) diffraction peaks smaller, as shown in The diffraction peaks’ shift indicated that the lattice parameters of their corresponding phases also changed. The lattice parameters of composites were calculated by the XRD patterns . The latβ-ZrSi (CrB-type structure) exists above 1460 °C, and transitions to α-ZrSi (FeB-type structure) occur below this temperature according to the Si-Zr phase diagram [β-ZrSi was found in the Z50T20S sample at room temperature, and its (002) diffraction peak shifted from 48.5 to 49.5 compared to the PDF card (# 04-004-7165), as shown in β-ZrSi lattice to form a Si solid solution (CrB-type) and inhibited the transition from the β to the α. diagram . Neverth0.5N0.5, and Si during sintering, the equilibrium phase diagram was calculated using FactSage software  and solid solutions. However, the first stage is still dominated by the reaction because of the low temperature. Therefore, TiC0.5N0.5 reacts with Si at this stage based on our previous research [Simultaneously, interdiffusion between ZrC and TiCresearch , as show2 forms the Ti-Si liquid phase as the temperature reaches 1330 °C [α-ZrSi transforms into β-ZrSi at 1460 °C [β-ZrSi can dissolve in Ti-Si liquid to form the Zr–Ti–Si liquid phase. The carbon atoms in the ZrC matrix diffuse into the Zr–Ti–Si liquid phase as the temperature increases, forming SiC, ZrC1−x, and TiC1−x [1−x and TiC1−x in the second stage gradually dissolve into the ZrC and TiC0.5N0.5 lattices and finally form  and solid solutions (ss), as shown in Equation (3).Second, the eutectic reaction of Si and TiSi 1330 °C . Then, td TiC1−x . In the β-ZrSi during the cooling process. Therefore, β-ZrSi was found in the Z50T20S specimen.Compared with the other two specimens, the Si content in Z50T20S reached 20 mol%, causing the liquid phase of Zr-Ti-Si generated in the second stage to not entirely participate in the reaction, and the liquid phase residual transitioned to 0.5N0.5 addition. As shown in 2 region” on the ZrC-TiC0.5N0.5-Si phase diagram. Therefore, the above phenomenon may be related to the appearance of N2. Similar phenomena have also occurred in other studies. Liang et al. [0.5N0.5 becomes unstable at higher temperatures and denitrifies to produce N2. Denitrification is worse when the amount of nitrogen and temperature are both high. The generated N2 will eventually form pores in the ceramic matrix at high sintering pressure. The minimum RD of the prepared TiC0.5N0.5 ceramics was 94.3%, consistent with the above analysis. As shown in 0.5N0.5 is not sensitive to denitrification at 1500–1700 °C [The curves of relative densities and grain sizes versus Si content for the prepared composites are shown in g et al.  prepared–1700 °C ,18. Mean–1700 °C . Thus, u0.5N0.5 sintered at 2100 °C had more surface porosity and poorer densification. Like pure ceramics, the Z50T sample exhibited high porosity, as shown in β-SiC, respectively. Likely attributable to the influence of solid solution factors, the contrast of  and β-ZrSi was close (see the sequent HAADF images), so it could not be clearly distinguished on the BSE image of the Z50T20S sample.SEM micrographs of sintered ceramics are shown in 0.5N0.5-Si system [β-ZrSi does not exist in the Z50T5S ceramic, which has a phase composition of -31.2 vol% -4.2 vol% SiC. For the Z50T10S sample, the existence of β-ZrSi needs to be further analyzed using TEM. The contents of  and  solid solutions gradually decrease with increasing Si addition in -based composites. The average grain size of each phase in the sintered ceramics is shown in i system . Therefoβ-ZrSi. This indicated the existence of traces of β-ZrSi in the Z50T10S sample. According to the analysis of the reaction process in The Z50T10S was characterized by STEM to investigate the microstructure in detail. The HAADF image with element distribution maps and EDS spectra are shown in 0.5N0.5 lattice were 28.90% and 8.15%, respectively. However, the solubility of Ti atoms in the β-ZrSi lattice is detected at 10.81%. It was revealed that the Ti atoms present in the β-ZrSi lattice could stabilize the high-temperature phase at room temperature. β-SiC, respectively. SiC and ZrSi grains were surrounded by larger  and  grains, which formed a typical shape in-situ reaction. The grain sizes of , , and SiC  solid solution appeared in the matrix compared with the Z50T. Furthermore, the lower sintering temperature resulted in lower solid solubility. Z50T5S has a lower hardness than Z50T, with a hardness value of 24.5 ± 0.7 GPa. As the sintering temperature was further reduced, the hardness of Z50T10S and Z50T20S decreased. However, since the hardness of ZrSi was approximately 10 GPa [The mechanical properties of monolithic ZrC, TiCN, and ZrC-SiC, and the ZrC-TiCN composites examined by the authors in ,40,41,425–29 GPa . This suy 10 GPa , the har0.5N0.5 system, the ceramics possessed a higher strength. The flexural strength of Z50T10S reached a value of 435 ± 28 MPa, which was higher than that of  ceramics, as shown in σ, ICK, Y, and a are the flexural strength, fracture toughness, geometric constant, and defect size of ceramic, respectively. Grain size is usually considered the characteristic critical defect size in almost completely dense ceramics. Thus, the higher flexural strength of ceramics with the Si addition may be correlated with the higher fracture toughness. Z50T5S exhibited a high RD and fine grain size, with a flexural strength of 508 ± 33 MPa, representing the maximum in prepared ceramics. Compared to ZrC-SiC ceramics [0.5N0.5, and ZrSi were 4.5 × 10−6, 6.8 × 10−6, 7.4 × 10−6, and 8.6 × 10−6 K−1, respectively [The flexural strength of Z50T was 297 ± 49 MPa lower than that of pure ZrC and   ceramicsceramics ,40,41, Zectively ,45. Cons1/2. With the Si addition increased, the fracture toughness of composites (20 mol% Si addition) increased to 4.4 ± 0.2 MPa·m1/2, caused by a significant rise in the fraction of the dispersed second phase in the matrix [The content of  solid solution in Z50T ceramics is much higher than that of  solid solution, so  can be regarded as the second phase, similar to SiC and ZrSi. Compared with pure ZrC and TiCN ceramics, the fracture toughness of Z50T was improved to 3.3 ± 0.1 MPa·me matrix . As showe matrix . Under t0.5N0.5, and Si powders as the raw materials. The addition of Si significantly reduced the sintering temperature due to the liquid phase formation during sintering. At the low sintering temperature, both  and  solid solutions appeared in the composites, and the mechanical properties were enhanced by adjusting the ratio of ZrC to TiC0.5N0.5. The liquid phase generated and the  and  solid solutions were conducive to facilitating the mass transfer and promoting densification. During the sintering process, all of the Si that was added in a 5 mol% amount turned into SiC. With the increased Si addition, the residual ZrSi phase appeared, reducing the flexural strength and hardness of composites while improving their toughness.  and SiC effectively inhibited the growth of matrix grains. The -based composite with 47.5 mol% ZrC, 47.5 mol% TiC0.5N0.5, and 5 mol% Si possessed excellent comprehensive mechanical properties . The higher flexural strength and hardness were mainly attributed to low porosity, refined grains, solid solution strengthening, and second-phase strengthening; meanwhile, the crack deflection and bridging caused by the  and SiC particles resulted in higher toughness.We produced almost completely dense -based nanocomposites via reactive hot pressing at 1500–1700 °C using ZrC, TiC


Background and Objectives: Faricimab is a novel bispecific antibody with Fab regions inhibiting both vascular endothelial growth factor-A and angiopoietin-2. Therefore, this study aimed to obtain short-term outcomes of intravitreal injection of faricimab (IVF) for the treatment of diabetic macular edema (DME) in daily clinical practice. Materials and Methods: A retrospective review was carried out on consecutive patients with DME who had been treated with IVF and were followed up for at least 1 month. Outcome measures included changes in logMAR best-corrected visual acuity (logMAR BCVA), central retinal thickness (CRT), number of IVF administrations, and safety. Clinical outcomes were also compared between the treatment-naïve and switch groups. Results: A total of 21 consecutive DME eyes from 19 patients were identified. The mean number of IVFs was 1.6 ± 0.8 during the mean follow-up time of 5.5 months. The overall mean logMAR BCVA following IVF was 0.236, 0.204, 0.190, and 0.224 at baseline, 1, 3, and 6 months, respectively, without a significant change from baseline to 1 month (p = 0.176) or for 6 months (p = 0.923). The overall mean CRT (μm) following IVF was 400.6, 346.6, 342.1, and 327.5 at baseline, 1, 3, and 6 months, respectively. CRT significantly decreased from baseline to 1 month (p = 0.001) but did not reach a significant level over 6 months following IVF (p = 0.070). No significant difference in BCVA or CRT was observed between the treatment-naïve and switch groups. No serious safety concerns were noted. Conclusions: IVF for the treatment of DME may preserve visual acuity and improve macular thickness without serious safety concerns in the short term in a real-world clinical setting. Previous basic studies clearly revealed that vascular endothelial growth factor-A (VEGF-A) and angiopoietin 2 (ANG2) are major players in the pathogenesis of diabetic macular edema (DME) . HoweverFaricimab  is a bispecific antibody with Fab regions binding to VEGF-A and ANG2 to act as both VEGF-A and ANG2 signal inhibitors. Based on the successful results of two multicenter phase 3 randomized clinical trials (RCTs) (YOSEMITE and RHINE), faricimab was approved and launched as a treatment for DME in 2022. YOSEMITE and RHINE trials demonstrated non-inferior vision gains and favorable anatomical outcomes in both faricimab every 8 weeks and personalized treatment interval (adjustable up to 16 weeks) arms as compared to aflibercept every 8 weeks arm at 1 year, indicating that patients with DME treated with faricimab might achieve optimal treatment outcomes even if the dosing interval is extended . HoweverWe retrospectively reviewed the medical records of consecutive patients with DME who had been treated with IVF at Kobe University Hospital and followed up for at least 1 month. Approval for this study was granted by the institutional review board of the Kobe University Graduate School of Medicine (permission number: 170064). This study adhered to the tenets of the Declaration of Helsinki (7th revision) for research on human subjects, and the IRB waived obtaining informed consent from patients due to the retrospective nature of this study. However, patients were provided the opportunity to express their choice of data used by an opt-out system through the hospital’s website.The following data were collected for analyses: age, sex, eye laterality, axial length, lens status, previous vitreous surgery, decimal best-corrected visual acuity (BCVA) , central retinal thickness (CRT) as determined by Macular Cube 200 × 200 scan data acquired by optical coherence tomography  , type of therapy (treatment-naïve or switch), the number of IVFs, type, and number of another treatment, follow-up period, and adverse events.Intravitreal faricimab injection (IVF) was conducted on an outpatient basis. Under ocular surface anesthesia with 4% lidocaine eyedrops, eyelid skin and ocular surface disinfection was performed using 5% povidone-iodine and 8-fold diluted PA IODO Ophthalmic and Eye washing Solution Disinfection . An eyelid speculum was then placed to keep the eye open, and faricimab (6 mg/0.05 mL)  was intravitreally injected using a 30-G needle (1/2 inch in length). Thereafter, antibiotic eyedrops and ointment were administered, the eyelid speculum was removed carefully, and an eyepatch was applied. The patient was instructed to remove the eyepatch the next day and continue antibiotic instillation (4 times daily) for 4 days.The primary outcome was set as logMAR BCVA changes from baseline to 1 month after the first IVF, and secondary outcomes were logMAR BCVA changes at and after 2 months from baseline; CRT changes from baseline to each time point after the first IVF, and the number of IVFs, additional treatments, and complications/side effects during the follow-up period. The baseline value was defined as the nearest time point before the first IVF.p-values of <0.05 were considered significant.Continuous variables were provided as mean ± standard deviation unless otherwise specified. Missing follow-up visit values were imputed using the last observation carried forward method for statistical analyses as needed. The generalized liner-mixed model was performed in the statistical analyses of changes among different time points using a graphical user interface for R software  and EZR . For other statistical analyses, MedCalc v.20.027 software  was used. A total of 21 consecutive eyes from 19 patients were identified as fulfilling the inclusion criteria. The mean age was 67.7 ± 7.2 years; four patients (21%) were females, and five eyes (24%) had a history of prior vitrectomy. Among the 21 eyes, 14 (67%) were treatment-naïve before IVF, and the distribution of diabetic retinopathy severity was 19%, 71%, and 10% for moderate nonproliferative diabetic retinopathy (moderate NPDR), severe nonproliferative diabetic retinopathy (severe NPDR), and proliferative diabetic retinopathy (PDR), respectively. The mean follow-up time was 5.5 ± 2.0 months, and 1 case (5%) had a follow-up period of only 1 month. The summary of patients’ characteristics is shown in n = 7), intravitreal ranibizumab (0.5 mg) injection (n = 1), and macular photocoagulation (n = 1).In the switch group, treatments performed within 6 months before the first IVF were intravitreal aflibercept (2 mg) injection (pro re nata: PRN). During the mean follow-up time of 5.5 months, the mean number of IVF was 1.6 ± 0.8 , and 12 cases (57%) had only 1 faricimab injection. The vitrectomized eyes had received more IVF than the non-vitrectomized eyes . Of 21 eyes, 12 cases (57%) had only one faricimab injection, and 15 eyes (71%) continued the IVF treatment; however, the remaining six eyes (29%) switched to other treatments: intravitreal aflibercept (2 mg) injection (n = 2), intravitreal brolucizumab (6 mg) injection (n = 2), sub-Tenon’s injection of triamcinolone acetonide (n = 2), and pars plana vitrectomy (n = 1). The treatments were switched because of insufficient CRT decrease in two eyes, CRT worsening in two eyes, and faricimab side effects in two eyes. Of six switched cases from IVF, four cases (67%) were treatment-naïve, and two cases (33%) were switched cases at baseline.The scheme of the intravitreal injection was as needed (p = 0.176) or over 6 months following the IVF (p = 0.923). The mean logMAR BCVA in the treatment-naïve group was 0.234 ± 0.264, 0.205 ± 0.296, 0.209 ± 0.290, 0.183 ± 0.320, 0.224 ± 0.325, 0.219 ± 0.336, and 0.223 ± 0.324 at baseline, 1, 2, 3, 4, 5, and 6 months, respectively, whereas the mean logMAR BCVA in the switch group was 0.241 ± 0.209, 0.200 ± 0.175, 0.192 ± 0.178, 0.203 ± 0.183, 0.220 ± 0.230, 0.234 ± 0.238, and 0.227 ± 0.244 , although the CRT change did not reach a significant level over 6 months following the IVF (p = 0.070). The mean CRT (μm) in the treatment-naïve group was 394.2 ± 112.1, 357.4 ± 100.6, 355.1 ± 103.0, 348.1 ± 118.2, 333.1 ± 106.5, 333.1 ± 107.9, and 354.6 ± 107.2 at 6 months, whereas the CRT (μm) in the switch group was 413.4 ± 60.8, 325.0 ± 51.5, 369.1 ± 149.2, 330.3 ± 49.0, 323.1 ± 65.7, 298.1 ± 62.6, and 273.4 ± 54.3 at baseline, 1, 2, 3, 4, 5, and 6 months, respectively  following the IVF was 400.6 ± 96.8, 346.6 ± 87.3, 359.8 ± 116.7, 342.1 ± 99.4, 329.8 ± 94.3, 320.3 ± 93.8, and 327.5 ± 99.4 at baseline, 1, 2, 3, 4, 5, and 6 months, respectively. CRT significantly decreased from baseline to 1 month , epilepsy (n = 1), and anterior uveitis (n = 1). The eyes with macular edema deterioration were successfully treated with either intravitreal brolucizumab (6 mg) injection or pars plana vitrectomy. Epilepsy healed spontaneously, and anterior uveitis rapidly subsided with 0.1% fluorometholone eyedrops.Although no intravitreal injection-related complications  were noted during the follow-up period, side effects in which a relationship to the administered drug (faricimab) cannot be denied were observed: worsening of macular edema  had a CRT of <325 μm at 1 month, and the proportion of eyes with CRT of <300 μm was 38% at 1 month and 71% at the final visit. CRT results in our study are non-inferior to those in YOSEMITE/RHINE trials and Rush’s study. In YOSEMITE/RHINE trials, approximately 40% of eyes attained complete disappearance of DME (defined as CRT <325 μm) at 1 month . In compRegarding the safety of IVF, we did not encounter any unexpected adverse events. Although a patient with epilepsy was examined after being hospitalized, the internist in charge believes that epilepsy was caused by systemic background. Accordingly, IVF for the treatment of DME is likely well tolerated in our study.Most of the limitations of our study stem from the small number of cases which precluded us from performing detailed analyses on the true effects of treatment-naïve and switch groups, history of prior vitrectomy, treatment regimen on clinical outcomes, and safety. For example, our preliminary analysis showed that vitrectomized eyes tended to have more IVF than non-vitrectomized eyes. However, we do not know whether this can be generalized, as the number of vitrectomized eyes was too small. Although short-term outcomes of the IVF in the switch group help consider a direct contribution of ANG2 to DME pathology for each eye, the long-term effects of dual inhibition of VEGF-A and ANG2 in real-world clinical settings should be clarified in the future. We understand that the evidence level of this study is not high. However, given the paucity of real-world data on IVF for DME at the moment, we hope that short-term real-world data in this study will be of some help.IVF for the treatment of DME may preserve visual acuity and improve macular thickness without serious safety concerns in the short term in a real-world clinical setting, which seems reasonable because both previous basic studies and recent clinical studies indicate the importance of VEGF-A and ANG2 in the pathogenesis and therapeutic targets of DME ,10,14,15


Fibrates are widely used hypolipidaemic agents that act as ligands of the peroxisome proliferator-activated receptor α (PPARα). p38 is a protein kinase that is mainly activated by environmental and genotoxic stress. We investigated the effect of the PPARα activators fenofibrate and WY-14643 and the PPARα inhibitor GW6471 on the levels of activated p38 (p-p38) in the colorectal cancer cell lines HT-29 and Caco2 in relation to their differentiation status. Fibrates increased p-p38 in undifferentiated HT-29 cells, whereas in other cases p-p38 expression was decreased. HT-29 cells showed p-p38 predominantly in the cytoplasm, whereas Caco2 cells showed higher nuclear positivity. The effect of fibrates may depend on the differentiation status of the cell, as differentiated HT-29 and undifferentiated Caco2 cells share similar characteristics in terms of villin, CYP2J2, and soluble epoxide hydrolase (sEH) expression. In human colorectal carcinoma, higher levels of p-p38 were detected in the cytoplasm, whereas in normal colonic surface epithelium, p-p38 showed nuclear positivity. The decrease in p-p38 positivity was associated with a decrease in sEH, consistent with in vitro results. In conclusion, fibrates affect the level of p-p38, but its exact role in the process of carcinogenesis remains unclear and further research is needed in this area. Fibrates, such as fenofibrate, bezafibrate, clofibrate, and gemfibrozil, are widely used hypolipidaemic agents. They are generally well-tolerated drugs with a good safety profile ,2,3. TheIn addition to exogenous ligands, PPARα is also activated by endogenous molecules such as dietary fatty acids, including arachidonic acid and its metabolites ,10,11. AThe intestinal epithelium is one of the most rapidly renewed tissues in the organism, with a turnover of approximately 5 days . Colorecp38 is mainly activated by environmental and genotoxic stress and is known as a stress-activated protein kinase. The four p38 MAPKs, namely p38α, p38β, p38γ, and p38δ, are encoded by different genes. p38α and p38β are ubiquitously expressed, but the expression of p38β is much lower than that of p38α. The expression patterns of p38γ and p38δ are more restricted. p38 activation occurs through dual phosphorylation of Thr and Tyr. p38 regulates multiple cellular functions including cell proliferation, differentiation, stress response, apoptosis, cell migration, and survival. The exact role of p38 in carcinogenesis is not fully understood. On the one hand, it acts as a tumour suppressor by promoting cell cycle arrest, cell differentiation, senescence, and apoptosis. On the other hand, p38 promotes cancer by increasing survival, migration, or resistance to stress and chemotherapeutic agents in tumour cells. The role of p38 depends not only on the type of stimulus, but also in a cell type-specific manner and on the level of activation, as strong and sustained p38 activation has been linked to apoptosis, senescence, and terminal cell differentiation, whereas low p38 activation has a cell survival effect ,25,26. pThere is limited information on the effect of PPARα ligands on p38. Fenofibrate has been shown to upregulate phosphorylated p38 (p-p38) in B16-100 melanoma cells, thereby inhibiting melanin synthesis . IncreasThe aim of this study was to investigate the effect of the PPARα activators fenofibrate and WY-14643 as well as the PPARα inhibitor GW6471 on the levels of p-p38 . Two colorectal carcinoma cell lines, HT-29 and Caco2, were used as models. We also investigated p-p38, CYP2J2 and sEH in human grade 2 colorectal carcinoma tissues and adjacent normal tissues.p = 0.0106) and 193.60 ± 55.34 (p = 0.0020) % of control, respectively. WY-14643 at concentrations of 25 μM and 200 μM increased p-p38 expression to 190.50 ± 80.48 (p = 0.0155) and 201.30 ± 116.50 (p = 0.0434) % of control, respectively. The PPARα inhibitor GW6417 at concentrations of 1 μM and 10 μM increased p-p38 expression to 149.80 ± 41.85 (p = 0.0120) and 168.70 ± 53.33 (p = 0.0082) % of control, respectively.Changes in p-p38 expression were assessed by ICE see , parts Ap < 0.0001) and 84.63 ± 14.32 (p = 0.0466) % of control, respectively. WY-14643 at 25 μM and 200 μM reduced p38 expression to 63.80 ± 16.57 (p = 0.0222) and 76.44 ± 19.01 (p = 0.0286) % of control, respectively. Treatment with 1 μM and 10 μM GW6471 resulted in a slight non-significant increase in p-p38 expression to 112.40 ± 37.37 (p = 0.4545) and 125.00 ± 57.42% (p = 0.2577) of control, respectively.In contrast to undifferentiated HT-29 cells, we observed a significant decrease of p-p38 in sodium butyrate-differentiated HT-29 cells after treatment with PPARα activators. Fenofibrate at concentrations of 25 μM and 150 μM decreased p-p38 expression to 48.00 ± 14.72 (p = 0.0127) and 55.66 ± 12.29 (p = 0.0003) % of control, respectively. WY-14643 at concentrations of 25 μM and 200 μM decreased p-p38 expression to 57.88 ± 19.30 (p = 0.0005) and 45.30 ± 37.85 (p = 0.0087) % of control, respectively. Treatment with 1 μM and 10 μM GW6471 resulted in non-significant changes in p-p38 expression compared to control: 121.70 ± 67.05 (p = 0.3591) and 84.61 ± 55.07% (p = 0.4551), respectively.In undifferentiated Caco2 cells, we observed a significant decrease in p-p38 after treatment with PPARα activators. Fenofibrate at concentrations of 25 μM and 200 μM decreased p-p38 expression to 55.07 ± 45.82 (p = 0.0010) and 54.63 ± 31.45 (p = 0.0025) % of control, respectively. WY-14643 at concentrations of 25 μM and 200 μM decreased p38 expression to 46.82 ± 38.97 (p = 0.0380) and 65.89 ± 34.83 (p = 0.0416) % of control, respectively. Treatment with 1 μM and 10 μM GW6471 resulted in a slight, non-significant increase in p38 expression to 112.10 ± 17.33 (p = 0.1143) and 114.90 ± 15.58% (p = 0.1526) of control, respectively.In spontaneously differentiated Caco2 cells, we observed a significant decrease in p-p38 after treatment with PPARα activators. Fenofibrate at concentrations of 25 μM and 200 μM decreased p-p38 expression to 48.73 ± 22.97 . We also found that differentiated HT-29 cells and undifferentiated Caco2 cells differed significantly in villin expression .Because of the opposite response in p-p38 levels of undifferentiated and differentiated HT-29, undifferentiated Caco2 and differentiated Caco2 to PPARα ligands treatment, we examined the differentiation status of HT-29 and Caco2 cells see . Villin p < 0.0001). In contrast, sEH expression is higher in undifferentiated Caco2 than in undifferentiated HT-29 cells (p = 0.0279). Both enzymes are increased in differentiated cells compared to undifferentiated cells in both cell lines. The increase of p-p38 in undifferentiated Caco2 compared to undifferentiated HT-29 cells approached significance with p = 0.0990. p-p38 is also increased in differentiated compared to undifferentiated cells in both cell lines.CYP2J2 expression is lower in undifferentiated Caco2 cells compared to undifferentiated HT-29 cells (p < 0.0001). In contrast, in colorectal carcinoma, p-p38 expression was higher in the cytoplasm than in the nucleus . Nuclear positivity of p-p38 was significantly higher in normal epithelium than in carcinoma  whereas cytoplasmic immunostaining was stronger in carcinoma than in normal epithelium (p = 0.0116). The expression pattern of p-p38 in tumours was not affected by sex, tumour size, or lymph node infiltration.We found significant differences between the immunostaining intensity of p-p38 in the nucleus and cytoplasm in both normal adjacent colonic surface epithelium and in colorectal carcinomas see . In normp = 0.0015). In contrast to p38 and sEH, CYP2J2 immunostaining intensities were comparable in normal adjacent tissue and colorectal carcinoma (p = 0.3984).Immunostaining of sEH was significantly decreased in carcinoma compared to normal epithelium  in the colorectal cancer cell lines HT-29 and Caco2 cells in relation to their differentiation status. We also investigated p-p38, CYP2J2 and sEH in human grade 2 colorectal carcinoma tissues and adjacent normal tissues.Fenofibrate and WY-14643 affected p-p38 levels in both undifferentiated and differentiated cells of both cell lines. The PPARα inhibitor affected p-p38 levels only in undifferentiated HT-29 cells. Surprisingly, undifferentiated HT-29 cells showed an opposite response to PPARα activators treatment than the rest of the cells tested, namely, differentiated HT-29 as well as Caco2 cells regardless of their differentiation status. Fibrates increased p-p38 in undifferentiated HT-29 cells, whereas in other cases p-p38 expression was decreased.It is known that p-p38 phosphorylates substrates both in the cytoplasm and in the nucleus. In the cytoplasm, p38 can phosphorylate other kinases such as MNK1/2, while in the nucleus it activates transcription factors such as ATF2, p53 and STAT1 . HT-29 aThe different response of p-p38 expression to PPARα ligands could be influenced by the differentiation status of the cells. Although HT-29 and Caco2 cell lines are both derived from grade 2 colorectal adenocarcinomas, there are differences between them as they carry different mutations: HT-29 in p53 and BRAF, while Caco2 in APC, p53 and SMAD4 ,42,43,44As mentioned above, EETs are generally cytoprotective molecules with proliferative and anti-apoptotic functions ,13. EETsAlthough fibrates could affect p-p38 levels, it remains questionable whether this phenomenon is PPARα-dependent. It has been described that the PPARα activators fenofibrate and WY-14643 upregulated p38 phosphorylation in melanoma cells  and in aThe stage of tumour development may be an important determinant of the function of p38 in tumourigenesis. In general, low p38 activity impairs tumour formation and growth in early stages of the disease, while more advanced tumour stages may benefit from higher activation of the pathway . In coloTo investigate whether EETs might be related to p-p38 levels in colorectal carcinoma, we examined tissue samples by immunohistochemistry. We examined only grade 2 colorectal carcinomas so in terms of degree of differentiation it is a homogeneous group of samples. We found significant differences in the subcellular localisation of p-p38 compared to normal adjacent colon tissue. In carcinomas, higher levels of p-p38 were detected in the cytoplasm, whereas in normal colonic surface epithelium p38 was nuclear positive. The previous study by Gulmann et al. described that the activated isoform of p38 was decreased in colorectal carcinoma compared to the corresponding normal mucosa , which iAlthough it has been reported that CYP2J2 expression is higher in colorectal carcinoma than in normal adjacent tissue , we founIn the current study, we used colorectal carcinoma cell lines derived from moderately differentiated carcinomas (grade 2) as well as patient tissue samples of the same grade. This approach enables consistent data to be obtained. According to our results, the effect of fibrates on p-p38 levels depends on the differentiation status of the intestinal cells. Thus, additional useful information could be obtained by using patient samples from well-differentiated (grade 1) and poorly-differentiated (grade 3) tumours.In conclusion, fibrates increased p-p38 in undifferentiated HT-29 cells, whereas in other cases p-p38 expression was decreased. HT-29 cells showed p-p38 predominantly in the cytoplasm and PPARα ligands affected its expression in this cellular compartment, whereas Caco2 cells showed higher nuclear positivity and fibrates affected p-p38 expression in both compartments. Based on the expression of villin, p-p38 and sEH, it can be assumed that differentiated HT-29 and undifferentiated Caco2 cells share similar characteristics. Thus, the effect of fibrates may depend on the differentiation status of the cell. In colorectal carcinomas, higher levels of p-p38 were detected in the cytoplasm, whereas in normal colonic surface epithelium p-p38 showed nuclear positivity. The decrease in p-p38 positivity was associated with a decrease in sEH. Based on current knowledge, it can be concluded that fibrates affect the level of p-p38, but its exact role in the process of carcinogenesis remains unclear, so further research in this area is needed.2 and passaged twice a week.The HT-29 and Caco2 cell lines are derived from human grade 2 colorectal carcinomas. Cells were cultured and treated under the same conditions and experimental scheme as described in our previous studies ,52. The Cells were seeded onto 96-well culture plates  at a density of 10,000 cells/well (HT-29) and 7000 cells/well (Caco2) for In-Cell ELISA and onto 8-well cell culture slides  at a density of 18,000 cells/well for immunocytochemical staining and allowed to adhere overnight. The cells were then treated with fenofibrate , WY-14643  or GW6471  as follows: 25 μM and 150 μM (HT-29) or 200 μM (Caco2) fenofibrate, 25 μM and 200 μM WY-14643, and 1 μM and 10 μM GW6471. Cells were incubated at 37 °C for 72 h. Stock solutions of PPARα ligands were prepared by dissolving in DMSO. Control cells were treated with corresponding concentrations of DMSO. The concentrations used were determined using the WST-1 proliferation assay . The results of this assay under the same experimental conditions were published in our previous studies ,52.Differentiated HT-29 cells were obtained by treatment with 5 mM sodium butyrate  for 72 h. Differentiated Caco2 cells were obtained by postconfluent growth for 14 days with growth medium changes twice a week . After dn = 8).Changes in the expression of proteins of interest were assessed using the In-Cell ELISA colourimetric kit . The procedure was performed according to the manufacturer’s protocol. After the incubation period, cells were washed in PBS and fixed with 4% paraformaldehyde for 10 min at RT. Prior to primary antibody incubation, cells were permeabilised with 0.01% Triton-X100 (included in the kit) and pretreated with 1% hydrogen peroxide . The samples were incubated overnight at 4 °C with the following primary antibodies: phospho-p38 MAPK alpha   at 1:250 dilution, villin  at 1:1500 dilution, CYP2J2  at 1:100 dilution, EPHX2  at 1:50 dilution. The samples were then incubated with horseradish conjugate followed by TMB substrate (part of the In-Cell ELISA colourimetric kit). The measured antibody signals (absorbance at 450 nm) were normalised to whole cell staining using Janus Green (absorbance at 615 nm). Results are expressed as relative expression [%] compared to the corresponding control cells (100%). Absorbance was measured using a Power Wave XS microplate reader (Bio-Tek). The experiment was performed in four independent duplicates . After pre-treatment with PolyDetector Peroxidase Blocker  for 5 min and ProteinBlock  for 30 min, samples were incubated with primary antibody against phospho-p38 MAPK alpha   at 1:75 dilution for 1 h at RT. Visualisation was performed using the Mouse/Rabbit PolyDetector DAB HRP Brown Kit . Tris buffer with TWEEN 20 (pH 7.6) was used for washing between different steps. Cell nuclei were counterstained with hematoxylin. The intensity of immunostaining was evaluated separately in the cytoplasm and in the nucleus. The scoring was semiquantitative as follows: 0 for negative tissue, 1 for weak signal, 2 for moderate signal, and 3 for strong signal. The evaluation was performed in 5 different fields of vision at 200× magnification and the percentage of cells in each category was counted. Finally, the histoscore for each treatment was counted as: H = [(1 × %weakly positive cells) + (2 × %moderately positive cells) + (3 × %strongly positive cells)]. Thus, the maximal value of 300 could be reached.Basic patient characteristics of tissue samples from moderately differentiated (grade 2) colorectal adenocarcinoma and adjacent normal colonic tissue are shown in Antigens of interest were detected in 4 µm thick paraffin sections using the following primary antibodies: phospho-p38 MAPK alpha   at 1:75 dilution, CYP2J2  at 1:50 dilution, and sEH  at 1:200 dilution. After deparaffinisation and hydration, heat-induced antigen retrieval was performed in citrate buffer pH6 (CYP2J2 and sEH) or EDTA pH9 (p38) . The samples were then pretreated with PolyDetector Peroxidase Blocker  for 5 min and with ProteinBlock (Dako) for 30 min. Slides were incubated with primary antibodies for 1 h at RT and the reaction was visualised using the Mouse/Rabbit PolyDetector DAB HRP Brown Kit . Tris buffer with TWEEN 20 (pH 7.6) was used for washing between different steps. Cell nuclei were counterstained with hematoxylin. Slides were then washed in tap water, dehydrated, and coverslipped.Staining intensity was scored semiquantitatively as follows: 0 for negative tissue, 1 for weak signal, 2 for moderate signal, and 3 for strong signal. Due to the subcellular localisation of the studied antigens, the staining of CYP2J2 and sEH was evaluated in the cytoplasm, whereas the staining of p38 was evaluated in the nucleus and cytoplasm separately. To prevent observer bias, the slides were coded before scoring and the slides were scored twice at different times.t-test against the control value of 100%. The results of immunohistochemical staining of tissue samples were evaluated by the Wilcoxon signed rank test. All calculations were performed using Graph Pad Prism 8 at a significance level of p < 0.05. Statistically significant differences are marked with an asterisk (*) directly in the graphs: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.The results obtained from the In-Cell ELISA were evaluated by one-sample


Background and objective: As a partial positive allosteric modulator of the gamma-aminobutyric acid A (GABAA) receptor, dimdazenil was used for the treatment of insomnia with the potential to alleviate associated side effects compared to full agonists. The objective of this trial is to assess the safety, tolerability, food effect and pharmacokinetics following single and multiple doses of dimdazenil in Chinese healthy subjects.Methods: In this phase 1 trial, 36 healthy subjects aged ≥18 years were assigned to receive a single dose of 1.5, 2.5, or 5 mg dimdazenil, with each dose cohort consisting of 12 subjects, and 14 subjects were assigned to receive a multiple 2.5 mg daily dose of dimdazenil for 5 days. Safety, tolerability, and pharmacokinetic characteristics were evaluated.Results: Of the 50 subjects enrolled and 49 completed the trial, the incidences of treatment-emergent adverse events (AEs) in the single-dose groups of 1.5, 2.5, and 5 mg were 16.7%, 58.3% and 66.7% respectively, while 61.5% in the multiple-dose group. There were no serious AEs, deaths, AEs leading to discontinuation or AEs of requiring clinical intervention in any treatment groups. The most treatment-emergent AEs were dizziness , hyperuricemia , upper respiratory tract infection , diastolic blood pressure decreased , blood TG increased  and RBC urine positive . All AEs were mild-to-moderate and transient, and no severe AEs were documented in any study phase. The PK profile of dimdazenil and its active metabolite Ro46-1927 was linear across 1.5–5 mg oral doses in humans. The median Tmax for dimdazenil was in the range of 0.5–1.5 h, and the apparent terminal t1/2z ranged from 3.50 to 4.32 h. Taking Dimdazenil with food may delay Tmax and decrease Cmax, without affecting the total exposure (AUC). No relevant accumulations of dimdazenil and Ro 46–1927 were observed in multiple-dose group.Conclusion: Dimdazenil was generally well tolerated in healthy Chinese subjects after single and 5 days-multiple dosing. The pharmacokinetic properties of dimdazenil are compatible with a drug for the treatment of insomnia.Clinical Trial Registration: chinadrugtrials.org.cn, identifier CTR20201978 As a common ailment among adults, insomnia affects up to 30% of people worldwide . About 1etc.), dual orexin receptor antagonists (DORAs), sedative antidepressants and melatonin receptor agonists (Current pharmacotherapies include gamma-aminobutyric acid A (GABAA) receptor positive allosteric modulators such as benzodiazepines (BZDs) or non-benzodiazepine drugs . Highly expressed in the cortex, the alpha-1 subtype probably mediates the sedative and hypnotic effects  included two phases, the fasted phase was followed by the fed phase. After a screening period, subjects of three dose groups  were treated with a single oral dose of dimdazenil capsule in the morning while in the fasted state. After the fasted phase study completion, participants in 2.5 mg dose group were administered 2.5 mg dimdazenil underfed after a washout period of 14 days. During this phase, a high-fat meal was provided in the morning, and dimdazenil capsules were administered 30 min after the beginning of the meal to investigate the effect of food on PK. Following drug administration, each PK sample was taken on Day 1 at the following time points: pre-dose, 15 min, 30 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12 h post-dose, and at 24, 36, 48 and 72 h post-dose.In the multiple-dose part (Part 2), subjects received a daily 2.5 mg dose of dimdazenil for 5 days in the evening (about 21:00). Plasma samples were collected on Day 1 at the following time points: 15 min, 30 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, and 12 h post-dose, and pre-dose in the evening of Day 2, 3, 4 and 5. After the last drug administration, PK samples were taken at 15 min, 30 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48, and 72 h.2 and a body weight of ≥50 kg (men) or 45 kg . Subjects were generally healthy at screening and/or prior to the initial administration of a trial drug, the intraocular pressure of both eyes was in the range of 10–21 mmHg, and the oxygen saturation of finger pulse blood was within the scope of 95%–100%. Concomitant medications were not permitted during the study, guaranteeing that the investigation of safety/tolerability and PK variables would not be affected by other drugs.A total of 50 healthy subjects included in this trial, 36 subjects were in Part 1 and 14 subjects were in Part 2. Eligible participants were aged ≥18 years with a body mass index (BMI) of 19–26 kg/mThe main exclusion criteria were a history of significant medical, neurologic, or serious psychiatric illness. Besides, any caffeinated, alcoholic xanthine-rich food or drink consumed within 48 h before dosing was not allowed.The study was conducted at Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine. The protocol was approved by the Center for Drug Evaluation, NMPA. Regulatory and institutional review board or independent ethics committee approval was obtained for each subject and written informed consent was obtained from all participants in accordance with local regulations.The objective of this study was to evaluate the safety, tolerability and PK characteristics of single- and multiple-dose oral administration of dimdazenil in healthy adults, as well as the effects of food on the PK of dimdazenil. In this study, participants were monitored as inpatients, with day −1 considered to be the day before initial administration. Participants were administered treatment on day 1, and assessed for safety/tolerance and PK characteristics at selected times during this study. Participants were considered to complete the trial according to the protocol after their final planned safety follow-up visit.max), time to reach the maximum observed concentration (Tmax), apparent terminal elimination half-life (t1/2z), an area under the concentration–time curve (AUC) from time of administration to 24 h (AUC0–24h), AUC from time of administration to infinity (AUC0-∞), AUC from time of administration to the last measurable concentration (AUC0-t), apparent clearance (CLz), and apparent volume of distribution (Vz). Multiple-dose PK parameters calculated for dimdazenil included a maximum observed concentration in steady-state , AUC during the dosing interval (AUC0-τ), accumulation ratio based on Cmax (Ra(Cmax)) and AUC0-τ (Ra(AUC)),AUC0-∞,ss, AUC0-t,ss, t1/2z, Tmax, CLz and Vz. PK parameters were also evaluated for Ro46-1927 and Ro18-5,528, the circulating metabolites of dimdazenil.Venous blood samples were taken pre-dose and at specified time points after drug administration. Evaluated single-dose plasma PK parameters for dimdazenil included maximum observed concentration , physical examination , intraocular pressure test, clinical laboratory tests , and 12-lead electrocardiography (ECG). Treatment-emergent adverse events (TEAEs) were recorded and analyzed based on their frequency, nature and intensity. Adverse events (AEs) were counted once per subject by coded preferred term, considering the worst severity and strongest causality. The severity grading utilized the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, and coding was performed with Medical Dictionary for Regulatory Activities (MedDRA), version 23.0. All reported AEs were assessed in terms of the occurrence time, the severity level, the duration, the measures taken, the ultimate outcome, and the prognosis.The participants who received at least one dose of dimdazenil were all included in the safety analysis. And the participants who received all planned doses of the regimen with valid post-drug concentration data were all included in the PK analysis.Plasma PK parameters were analyzed by WinNonlin software, version 8.1 or higher (Certara Institute), other analyses were performed using SAS software, versions 9.4 and higher (SAS Institute). The PK analysis utilized the typical non-compartmental approach, and the PK parameters were descriptively summarized by doses, including the number of observations, arithmetic mean, geometric mean, standard deviation (SD), coefficient of variation (CV), 95% confidence interval (CI) of the mean, minimum, quartile, median, maximum and quartile, as appropriate. Descriptive statistics were used to summarize demographic and baseline characteristics, including frequencies, percentages, means, SD, medians, quartiles, minimum values, and maximum values. Safety and tolerability results were analyzed descriptively by treatment cohorts.From 20 October 2020, to 14 December 2020, 50 healthy subjects were enrolled in the study, as shown in the trial flowchart in 2 (19.4–25.9 kg/m2). Demographic and baseline characteristics were similar between treatment groups.The characteristics of the participants at baseline according to the assigned groups are shown in p = 0.013), while there was no significant difference among the other single dose groups (p < 0.05).All reported TEAEs in this study are presented in The most frequent TEAEs were dizziness (8.2%), hyperuricemia (6.1%), upper respiratory tract infection (6.1%), DBP decreased (6.1%), blood TG increased (6.1%) and RBC urine positive (6.1%). All the participants who suffered events were eventually recovered/cured, in remission or stable. No evident changes in vital signs, laboratory findings, ECG values or intraocular pressure from which baseline, and none of the subjects had suicidal ideation or suicidal behavior reported during the study.The mean concentration-time profiles of dimdazenil and its circulating metabolites are depicted in max values of 0.75 (range 0.5–2), 0.75 (range 0.5–3.5), and 0.50 (range 0.5–5) h of 1.5, 2.5, and 5 mg, respectively, and the mean t1/2z values were 3.80 (1.40), 3.99 (1.16), and 4.32 (1.19) h. The mean Vz, and CLz were 67.93 (11.69) L and 14.26 (5.70) L/h in 1.5 mg group, 65.33 (8.78) L and 12.48 (4.78) L/h in 2.5 mg group, 71.10 (14.80) L and 12.61 (5.69) L/h in 5 mg group, which are similar among different dose groups. A summary of the single-dose PK parameters of metabolites Ro46-1927 and Ro18-5,528 is shown in After single dose administration, dimdazenil capsules were absorbed rapidly with median Tβ (two-sided 95% CI) of Cmax, AUC0-t and AUC0-∞ was 0.912 (0.741–1.083), 1.099 (0.802–1.396) and 1.085 (0.792–1.378), respectively. The estimated slope β (95% CI) of Cmax, AUC0-t and AUC0-∞ for Ro46-1927 was 0.883 (0.762–1.005), 1.125 (0.787–1.464), and 1.123 (0.781–1.465), respectively. The estimated slope β (95% CI) for of Cmax, AUC0-t and AUC0-∞ Ro18-5,528 was 0.777 (0.619–0.934), 0.817 (0.652–0.983), and 0.601 (0.208–0.994), respectively. The PK profiles of dimdazenil and Ro46-1927 were linear across 1.5–5 mg oral doses in humans, Cmax, AUC0-t and AUC0-∞ were increased proportionally to the dose since the two-sided 95% CIs for the estimated value of β contained 1, while Ro18-5,528 exhibited less-than-dose-proportional increases  and 1.05 (0.09), respectively. The accumulation ratios for AUC0-τ and Cmax of Ro46-1927 were 1.14 (0.13) and 1.15 (0.10), and those of Ro18-5,528 were 3.10 (0.38) and 6.05 (0.79)  . In bothmax, AUC, and Tmax geometric means for dimdazenil and its metabolites were compared under fed and fasted conditions to evaluate the potential impact of food. The fed/fasted geometric mean ratios (GMR) and 90% CI for Cmax, AUC0–t and AUC0-∞ were 75.86% , 98.13%  and 98.39% , respectively. The median of Tmax values (range) were 0.75 h (0.5–3.5 h) under fasted state and 2.50 h (1.5–5 h) under fed state. Following administration of a high-fat diet, dimdazenil exhibited a 24% decreased in Cmax, a 2% decreased in AUC, and a 2 h delay in Tmax. These findings indicated that food may delay the Tmax and decrease the Cmax, but it does not significantly affect the total exposure (AUC).The Cmax, AUC0–t and AUC0-∞ were 81.11%, 92.18%, and 92.42%, the median Tmax value under fed and fasted conditions were 2.50 h (range 1–8 h) and 4.00 h (range 2.5–12 h), respectively. Food consumption led to a delay in Tmax for Ro46-1927, with a 19% decreased in Cmax and an 8% decreased in AUC. Regarding Ro18-5,528, the fed/fasted GMR for Cmax and AUC0–t was 138.39% and 141.58%, the median of Tmax values under fed and fasted conditions were 30.00 h (range 24–48 h) and 36.00 h (range 24–48 h). After the diet, there was a delay in Ro18-5,528 Tmax, along with an approximately 38% increase in Cmax and a 42% increase in AUC0-t.For Ro46-1927, the fed/fasted GMR for CThe study provided important initial clinical pharmacology information about the PK and safety of dimdazenil in Chinese healthy subjects. The effects of food on a single dose of dimdazenil and metabolites were also studied in the trial.max and AUC values for dimdazenil increased with the dose, with the lowest values at 1.5 mg dose and the highest ones at 5 mg dose. The PK profiles of dimdazenil following single-dose administrations in the fasted state were characterized by absorption with a median Tmax ranging from 0.5–1.5 h, and a mean t1/2z ranging from 3.50–4.32 h across the dose groups. The elimination profiles suggested that dimdazenil could be sufficient to sustain sleep longer than recommended doses of short-acting Benzodiazepine receptor agonists (BzRAs) whose mean t1/2 were less than 3 h, but have a lower likelihood to induce residual effects than intermediate or long-acting BzRAs whose mean t1/2 were longer than 6 h  and was reached later (delayed Tmax by about 2.5 h) compared to the fasted conditions.These results suggest that, in order to achieve faster sleep onset, dimdazenil should not be administered with or immediately after a meal.According to the results of food and drug interaction study, it appeared that food had a moderate effect on the rate of absorption of dimdazenil. Under the fed conditions, the mean CIn terms of safety and tolerability, dimdazenil was generally well tolerated, AE occurrence appeared to be dose-dependent. There were no serious AEs or deaths, and no participants dropped out from the study prematurely due to the AEs. The majority of AEs reported were mild and resolved spontaneously, which recovered in all instances. Moreover, comprehensive safety assessments including vital signs, laboratory findings, ECG values or intraocular pressure did not reveal any significant changes compared to baseline measurements. Additionally, no instances of suicidal ideation, suicidal behavior or other mental health problem were reported during the study, suggesting that dimdazenil has a relatively low impact on psychiatric risks.The commonest AE across all groups was dizziness, reported on 8 occasions, 4 reported in 5 mg single-dose group and 4 reported in 2.5 mg multiple-dose group. Other central nervous system AEs related to benzodiazepine, including balance disorder, somnolence, headache and insomnia were not reported in this study. One subject occurred ocular hypertension (with the severity of grade I) in 2.5 mg single-dose cohort under the fed phase, which was eventually recovered without any processing, and further considered extraneous to dimdazenil by the investigator.Nevertheless, some limitations of the study should be considered carefully. First, the participants in this trial were healthy and aged ≥18 years, which may limit the safety and tolerance findings and PK analysis in elders and insomniacs. Second, another factor that may limit the interpretation of the results is that the proportionality for the metabolites has not been confirmed, which may affect the estimation of the correlation between AEs and metabolites. In addition, the efficacy and drug dependence of dimdazenil will be further investigated in phase 2 and phase 3 clinical trials.This phase 1 study exhibited that dimdazenil was generally well tolerated in healthy Chinese subjects at a single dose of 1.5–5 mg and multiple doses of 2.5 mg. This may be attributed to the partial GABAA agonist nature of dimdazenil, which theoretically lower the probability of some untoward effects relative to full agonists. The PK characteristics and safety findings in this study may imply that in the dose range of 1.5–5 mg, dimdazenil produce an effective function of sleep regulation for disorders in sleep initiation and sleep maintenance, with a low risk of side effects and residual sedation.