Pantoea, Bacillus, Paenibacillus, and Pseudomonas were isolated from the husk and endosperm of rice seeds. The strains Pantoea sp. S5-1, Pseudomonas sp. S5-38, and Pseudomonas sp. S7-1 were classified as the most promissory to increase rice growth as they demonstrated the presence of multiple plant growth-promoting traits such as the production of auxins, phosphate, and potassium solubilization, the production of siderophores, and the inhibition of the phytopathogen Pyricularia oryzae. The inoculation of strains of Pantoea sp. and Pseudomonas spp. in rice improves the height, root length, fresh weight, and dry weight of the shoot and root after 21 days post-inoculation in hydroponic assays. This study constitutes the first report on Cuban rice cultivars about the presence of endophytes in seeds and their potential to promote seedling growth. Pantoea sp. S5-1, Pseudomonas sp. S5-38, and Pseudomonas sp. S7-1 were selected as the more promising strains for the development of bio-stimulators or bio-inoculants for Cuban rice crops.Cuban rice cultivars INCA LP-5 and INCA LP-7 are widely distributed in Cuba and Caribbean countries. Although there are studies about rhizospheric bacteria associated with these cultivars, there are no reports about their seed-associated bacteria. This study aimed to isolate endophytic bacteria from rice seeds and select those with the greatest plant growth-promoting traits. A total of nineteen bacterial strains from the genera  Oryza sativa L.) is one of the most important crops worldwide. Its production reached around 519.5 million tons in the 2022–2023 season [Rice (Pyricularia oryzae and Tagosodes orizicolus [Polyphagotarsonemus latus (white mite) [Cuban rice cv. INCA LP-5 and INCA LP-7 are part of the eleven principal rice cultivars planted in Cuba . They hate mite) . Both cute mite) . TherefoThe use of plant growth-promoting bacteria (PGPB) is an environmentally friendly strategy for crop production . The benSphingomonas, Flavobacterium, Microbacterium, and Pseudomonas [Endophytic PGPB live asymptomatically inside vital plant organs and comprise bacteria acquired from seeds  or soils  ,14. Seedudomonas ,21. DespAzospirillum, Herbaspirillum, Bacillus, Burkholderia, Pseudomonas, and Azotobacter [Rhizobium, Enterobacter, Pantoea, Mitsuaria, Acinetobacter, and Pseudomonas were recently reported in the rhizosphere of Cuban rice cultivars INCA LP-5 and INCA LP-7 [In Cuba, the PGPB community associated with rice rhizosphere is mainly composed of species belonging to the genera tobacter ,23,24. MNCA LP-7 ,26. Howev/v) ethanol for 1 min and a calcium hypochlorite solution  for 20 min, while one gram of seeds without husk was surface-disinfected with 70% (v/v) ethanol for 5 min, kept in 6% (v/v) sodium hypochlorite for 15 min, and washed 20 times with sterile distilled water. The efficiency of superficial disinfection was assessed by placing 100 μL of the final rinse onto a tryptone–yeast extract (TY) medium  and a further 48 h of incubation at 30 °C. The absence of colonies was indicative of proper surface seed disinfection. Fresh certified rice cv. INCA LP-5 and INCA LP-7 seeds were acquired from the Basic Technological-Scientific Unit “Los Palacios” in Pinar del Rio, Cuba. One gram of seeds with husk was surface disinfected, according to García et al. , using 7−1 yeast extract, 0.5 g·L−1 protease peptone, 0.5 g·L−1 casein acidic peptone, 0.5 g·L−1 glucose, 0.5 g·L−1 soluble starch, 0.3 g·L−1 K2HPO4, 0.05 g·L−1 MgSO4*7H2O, 0.3 g·L−1 sodic pyruvate, 15 g·L−1 agar; pH 7.2) and a yeast–mannitol agar . Cultures were incubated for three days at 30 °C, and then colonies with different colors and morphologies were selected and purified. Isolates were stored at −20 °C with 20% (v/v) glycerol for further studies.To isolate endophytic bacteria, surface-disinfected seeds were macerated in a sterile mortar with 1 mL of a saline solution (0.9% NaCl). One hundred microliters of the suspension were cultured in three different media: TY, R2A . For isolate identification at the genera level, the closest type of strain was determined, and a phylogeny was constructed. The closest type strains were obtained by comparing the consensus sequences with the Ribosomal Database Project (RDP) . For phylogenetic analysis, the sequence alignments were performed using the ClustalW multiple sequence alignment tool in MEGA-X software (v. 10.0.4) [The bacterial isolates were identified by the sequence analysis of almost the entire e et al. . The uni 10.0.4) ,30. Phyl−1) was used as an inductor to synthesize indolic compounds. Production was detected as a pink coloration of the medium after 72 h of incubation at 30 °C. Gluconoacetobacter diazotrophicus strain Pal5 was used as a positive control [−1 of indolic compounds.Production of indolic compounds was assessed in triplicate using the Salkowski method (λ = 540 nm)  in a Var control . A posit−1 glucose, 2.0 g·L−1 yeast extract, 50 mL·L−1 of 10% K2HPO4, 100 mL·L−1 of 10% CaCl, 15.0 g·L−1 agar; pH 5.8) and on National Botanical Research Institute’s Phosphate (NBRIP) medium 2, 5.0 g·L−1 MgCl2*6H2O, 0.25 g·L−1 MgSO4*7H2O, 0.2 g·L−1 KCl, 0.1 g·L−1 (NH4)2SO4, 18.0 g·L−1 agar; pH 6.8) [−1 sucrose, 1.5 g·L−1 K2HPO4, 0.5 g·L−1 MgSO4*7H2O, 1.0 g·L−1 CaCO3, 15.0 g·L−1 agar; pH 7.5) was used to evaluate potassium phosphate solubilization [Pantoea sp. strain UYSB45 was used as a positive control [The phosphate solubilization capability of the isolates was evaluated in vitro. Ten microliters of a suspension of the optical density of 0.2 at 620 nm were spotted in triplicate in glucose a GL-rich medium supplemented with calcium phosphate (10.0 g·L pH 6.8) . Aleksanlization . A translization . Pantoea control .Herbaspirillum seropedicae strain Z67 was used as a positive control [The chromeazurol (CAS) agar assay was used to detect siderophore production . Aliquot control . w/v) Avicel, 0.2% (w/v) carboxymethyl cellulose, or 10% (w/v) of skimmed milk, respectively [Acinetobacter sp. strain UYSB41 was used as a positive control in hemicellulose and cellulose hydrolysis assays [Pseudomonas sp. strain UYFA214 was used as a positive control in protease activity assays [Hemicellulose, cellulose, and protease hydrolytic activities were evaluated with three replicates in plates containing a TY or TSA solid medium supplemented with 0.5% (ectively . Enzymats assays . Pseudomy assays .w/v) was added and then incubated for 20 min. The CV excess was removed with water, while the bound CV was released from the cells by adding ethanol. The suspension absorbance was measured at 570 nm using a VarioskanFlash v. 4.00.51 spectrophotometer (Thermo Scientific). Biofilm formation was detected using the crystal violet (CV) staining method. The isolates were grown with agitation in 96-well plates until an optical density of 0.2 at 620 nm. The plates were incubated for 48 h at 30 °C without agitation, and the supernatant was removed. Then, the plates were washed with phosphate-buffered saline, and the CV solution . Antagonism was evaluated in dual culture on a PDA medium , as previously reported [P. oryzae was evaluated after 13 and 16 days of incubation through the formula: PRGI = (1 − r1/r2) × 100, where PRGI is the percentage of radial growth inhibition of P. oryzae, r1 is the radial growth of P. oryzae in Petri dishes with bacteria strains, and r2 is the radial growth of P. oryzae in Petri dishes without bacteria strains [According to the screening of plant growth-promoting traits, the more promising bacterial strains were used to assess further antagonistic abilities against the fungal phytopathogen reported . Plates  strains . O. sativa cultivars INCA LP-5 and INCA LP-7 were surface-disinfected [w/v) agar in water and incubated in the dark at 30 °C for three days. Fifteen seedlings were sown into a (800 mL) pot  containing 50 mL of diluted Hoagland nutrient solution (1:2). Bacteria were grown in TY media at a concentration of 5 × 1010 CFU mL−1. Then, 0.5 mL of the inoculant was mixed with diluted Hoagland nutrient solution for a final concentration of 5 × 108 CFU mL−1 in each pot. A negative control without bacterial inoculation was included. Herbaspirillum seropedicae strain Z67 was used as a positive control [Seeds of infected . Surface control . The pots were covered with translucent nylon and were located in controlled conditions following a randomized design and using 30 seedlings per treatment (two pots with 15 seedlings per treatment). The seedlings were grown with a photoperiod of 12 h light/12 h darkness conditions at 26 °C/22 °C (day/night) and 70% relative humidity for 21 days, and at this moment, the seedlings were harvested. The height and root length (cm), shoot and root fresh weight (mg), and shoot and root dry weight (mg) (dried at 70 °C until constant weight) were determined.P. oryzae, and the rice growth in the hydroponic system were subjected to normality (Bartlett test) and variance homogeneity (Kolmogorov–Smirnov test). Simple classification analysis of variance (ANOVA) was applied, with the Tukey HSD mean comparison test for p < 0.05. Statgraphic Plus v. 5.0 software was used for statistical data processing, and Microsoft Excel 2010 software for its representation.Data from the screening for phosphate solubilizing abilities, percentage of radial growth inhibition of Pantoea and Pseudomonas  and 70% (seven strains) of the isolates from INCA LP-5 and INCA LP-7 seeds, respectively, were Gram-negative and belonged to the phylum Pseudomonadota. They were assigned to the genera udomonas . The 77.bacillus .Pantoea and formed a well-supported cluster related to some types of strains of Pantoea  in the root dry weight  in the rice seedlings cv. INCA LP-5 (Pseudomonas promote rice growth (Pseudomonas isolated from maize rhizospheres altered the root architecture and enhanced rice growth and yield [To know the effect of the more promising bacteria on plant growth, eight strains were selected and inoculated on Cuban rice cultivars. The application of seed endophytic bacteria for promoting plant growth is gaining importance due to endophyte beneficial effects and their environment. Despite some of the isolated bacterial genera that have been previously reported on promoting the seedling development of rice, this is the first time reporting seed endophytes from Cuban rice cultivars that promote plant growth. In this study, we highlight NCA LP-5 . The proe growth . Recentlnd yield ,66. DespPantoea sp. S5-1, Pseudomonas sp. S5-38, and Pseudomonas sp. S7-1 can be selected for the development of biostimulants aimed at enhancing rice crop growth. This study isolated and characterized endophytic bacteria associated with rice seeds, providing valuable insights into the bacterial communities linked to two of Cuba’s most significant rice cultivars. These results mark a pioneering investigation in the country, revealing the potential of nineteen strains as biofertilizers, biostimulants, and biocontrol agents, some of which effectively promote rice growth. This is of particular importance, considering that seed endophytes serve as the initial inoculum for the plant. Notably, strains like


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.  Betacoronavirus genus, which includes SARS-CoV and MERS-CoV (The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a major global health and economic crisis . SARS-CoMERS-CoV . Its genMERS-CoV . Due to MERS-CoV . The cleMERS-CoV .One of the least understood NSPs of coronaviruses is NSP15, a highly conserved nidoviral RNA uridylate‐specific endoribonuclease (NendoU), carrying the catalytic domain of the endonuclease family , 9. Nendde novo synthesized viral RNA  and hexameric (NendoUhex) forms. Combined structural and biochemical analyses indicate that NendoU activity depends on the hexamer dynamics and suggest that supramolecular interactions are involved in the enzyme regulation mechanism. Furthermore, we carried out a large-scale X-ray fragment screen to probe new putative druggable sites. Our data shed light on the understanding of NendoU enzymatic profiles and opens up the path for the development of new allosteric and competitive inhibitors.Here, we combine X-ray crystallography, cryo-electron microscopy (cryo-EM) and biochemical analysis to investigate the complex biochemical profile of SARS-CoV-2 NendoU in both monomeric (NendoUMT126808.1), which was generated using SCRIPT One-Step RT-PCR (Cellco Biotec) and random hexamer primers. The NSP15 coding sequence was amplified using FastPol (Cellco Biotec) with primers 5′-CAGGGCGCCATGAGTTTAGAAAATGTGGCTTTTAATG-3′ and 5′-GACCCGACGCGGTTATTGTAATTTTGGGTAAAATGTTTCTAC-3′, and inserted into pETM11/LIC (generously donated by Dr Arie Geerlof) using the Ligation-Independent Cloning method  supplemented with 0.1 mg/ml lysozyme, 10 U of benzonase (Cellco Biotec) and 1.0 mM dithiothreitol (DTT), and disrupted using sonication. Cleared lysate was passed through a 5 ml HisTrap HP column  equilibrated in Buffer A, and them protein was eluted with Buffer A supplemented with 300 mM imidazole. Excess imidazole was removed using a Sephadex XK 26/60 column  equilibrated with Buffer A. To obtain NSP15 in monomeric form (NendoUmon), the sample was incubated overnight at 8°C with 4 mM DTT and 0.1 mg/ml TEV protease, then passed through a 5 ml HisTrap HP column equilibrated in Buffer A to remove non-cleaved protein and TEV protease. To obtain NSP15 in hexameric form (NendoUhex), the 6×His-tag was not cleaved. For both forms, final purification was performed by size exclusion chromatography using a HiLoad Superdex 75 16/60 column  equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl, 5% (v/v) glycerol and 0.5 mM TCEP. Purity was confirmed by 12% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Protein concentration was estimated using the theoretical extinction coefficient at 280 nm of 33 140/M/cm (Transformed BL21(DE3) cells were growth in LB Lennox medium supplemented with kanamycin at 37°C until OD140/M/cm . Mutatiomon or 25 nM NendoUhex at 35°C. Time-course reactions were directly monitored in a Stratagene Mx3005P real-time PCR system (Agilent Technologies) using FAM filters. The initial velocity (v0) was estimated using the slope from linear regressions of the reaction monitored during minutes 3–10. All reactions were performed in triplicate, and reported data are the average and error bars the standard deviation (SD). Analysis and graphs were performed with OriginPro v9.0.Fluorogenic oligonucleotide substrate 5'6-FAM-dArUdAdA-6-TAMRA3' was purchased from GenScript. Unless stated otherwise, activity assays were performed in 25 mM Bis-Tris–HCl buffer pH 6.0, 0.25 μM substrate and 100 nM NendoU30-3' by nsp15, based on (The gel-based RNA cleavage assay was performed to verify the cleavage of the poly(U) biotinylated RNA substrate 5'-UGACCUCUAGCUAGAGGUCA(U)based on . A 15% ahex in experimental buffer were applied on the Quantifoil 300 mesh copper R1.2/1.3 grid. Buffers tested were: 50 mM Tris–HCl pH 7.5, 200 mM NaCl; 50 mM Bis-Tris–HCl pH 6.0, 200 mM NaCl; or 50 mM phosphate-buffered saline (PBS) pH 6.0, 200 mM NaCl. Samples containing oligo(dT) were made in 50 mM PBS pH 6.0, 200 mM NaCl buffer containing 5 μM oligo(dT)20 (Thermofisher™). The sample was blotted for 4 s  with a blot force of –12 and then plunge-frozen using a Vitrobot Mark IV (Thermo Fisher). Frozen samples were imaged on a Titan Krios (G2) microscope operated at 300 kV. Movies were collected on a K3 detector in super resolution counting mode with a slit width of 20 eV and at a nominal magnification of 105kx corresponding to a calibrated physical pixel size of 0.831 Å at the specimen level. Data acquisition was done using EPU software (version 2.6) with a defocus range of 1.4–3.2 nm. Data collection statistics are summarized in For sample preparation, 3 μl of 0.5 mg/ml NendoUab initio modeling and two rounds of heterogeneous refinement. Selected volumes were refined using Non-uniform Refinement and D3 symmetry. The detailed data processing flowchart schemes for samples in buffers 50 mM Tris–HCl pH 7.5 mM, 200 mM NaCl; 50 mM Bis-Tris–HCl pH 6.0, 200 mM NaCl; or 50 mM PBS pH 6.0, 200 mM NaCl are available in In summary, all movies were aligned using MotionCor2 , and proHelix processing was performed with relion v3.1 . Data wemon were obtained in multiple conditions. For that, the protein was concentrated to 3.4 mg/ml and crystallized in a sitting drop at 18°C by vapor diffusion. Hexagonal crystals containing a dihedral asymmetric unit were obtained in 15% polyethylene glycol (PEG) 8000, 0.1 M sodium/potassium phosphate pH 6.2 and 20% (w/v) PEG 3350, 100 mM Bis-Tris propane, pH 6.5, 200 mM sodium sulfate, and cryo-conditions were obtained by adding 20% (v/v) ethylene glycol. Orthorombic crystals containing a hexagonal asymmetric unit were obtained with protein incubated with 100 μM oligo(dT) and crystallized in 0.1 M trisodium citrate pH 5, 14% (w/v) PEG 6000, and cryo-conditions were obtained by adding 20% (v/v) ethylene glycol.Crystals of NendoUData were processed with XDS via autoPROC , 33, scamon at 3.4 mg/ml mixed with 300 nl of well solution containing 14% (w/v) PEG 6000, 0.1 M tri-sodium citrate pH 5.0 and 10 nl of seed stocks of the same condition in Swissci 96-Well 3-Drop plates (Molecular Dimensions) containing 30 μl of well solution. Crystals were formed after 4 days at 20°C by vapor diffusion. For soaking, 40 nl of each fragment compound from the XCHEM Poised Library ; ESIprot (Proteins for native MS were held on ice and buffer exchanged into 75 μl of 50 mM ammonium acetate pH 7.5 by three rounds of gel filtration using BioGel P6 (Biorad) spin columns according to the manufacturer's instructions. Mass spectra were acquired using an Agilent 6530 QTOF operating in positive ion 1 GHz mode using a standard ESI source. Samples were introduced via a syringe pump at a flow rate of 6 μl/min. The ion source was operated with the capillary voltage at 3500 V, nebulizer pressure at 17 psig, drying gas at 325°C and drying gas flow rate at 5 l/min. The instrument ion optic voltages were as follows: fragmentor 430 V, skimmer 65 V and octopole RF 750 V. The  ESIprot  and empl5′-Azido-5′-deoxy-thymidine (LIZA-7) was prepared according to the procedure we published recently  D = +40.9 ,1H NMR  δ (ppm) 7.45 , 6.16 , 5.43 , 4.11 , 3.80–3.71 , 3.23 , 3.11 , 2.37 , 2.28–2.18 , 2.06 , 1.81 . 13C NMR  δ (ppm) 194.9 , 163.7, 150.5 , 136.1 , 109.9 , 84.5, 83.9, 72.6 , 38.0 , 31.1 , 30.6 , 12.2 . MALDI-ToF MS: m/z calculated for C12H16N2NaO5S + [M + Na]+ 323.0672, found 323.0660.R5′-Deoxy-5′-thiothymidine (FÜZS-5): 5′-S-acetyl-5′-deoxy-5′-thiothymidine  was dissolved in dry MeOH (25 mL) under argon, and NaOMe  was added and stirred at room temperature for 4 h. The reaction mixture was neutralized with Amberlite IR 120 H+ ion exchange resin, filtered and evaporated under reduced pressure. The crude product was purified by flash column chromatography (CH2Cl2/MeOH 97.5/2.5→95/5) to give 5′-deoxy-5′-thiothymidine (FÜZS-5)  as a white solid.f = 0.20 (CH2Cl2/MeOH 95/5), m.p. 200–202°C; [α]D = +5.0 , 1H NMR  δ (ppm) 7.51 , 6.19 , 5.36 , 4.22 , 3.78 , 2.75 , 2.49 , 2.21 , 2.06 , 1.80 . 13C NMR  δ (ppm)163.7150.5 , 136.1 , 109.9 , 87.1 , 83.7 , 71.9 , 38.1 , 26.3 , 12.2 . MALDI-TOF MS: m/z calculated for C10H14N2NaO4S + [M + Na]+ 281.0566, found 281.0540.Rmon and NendoUhex were successfully purified as described. NendoUmon was obtained after cleavage of the N-terminal histidine tag as a single monodisperse peak by gel filtration , sodium cacodylate pH 6.0, 150 mM NaCl (v0 = 15.5 ± 0.08 RFU/min), MES pH 6.0, 150 mM NaCl (v0 = 16.5 ± 0.07 RFU/min) and Bis-Tris pH 6.5, 150 mM NaCl (v0 = 15.6 ± 0.14 RFU/min). A similar behavior was seen for NendoUhex, that presented maximum activity at sodium cacodylate pH 6.0, 150 mM NaCl (v0 = 998.4 ± 31.2 RFU/min) and Bis-Tris pH 6.5, 150 mM NaCl (v0 = 902.8 ± 13.90 RFU/min). For more basic buffers, such as MOPS pH 7.0, HEPES pH 7.5 and Tris pH 8.5, the enzyme activity of both NendoUmon and NendoUhex was virtually negligible. In PBS with 150 mM NaCl, NendoUhex activity was also significantly higher at pH 6.0 (v0 = 430.4 ± 15.0 RFU/min) than at pH 7.4 (v0 = 74.2 ± 3.0 RFU/min). Moreover, the overall reaction rate for NendoUhex was much higher than for NendoUmon for all tested conditions, similar to what has been reported previously . The t1/2 for temperatures of 25, 30, 35 and 40°C was 53.0, 29.7, 18.2 and 11.4 min, respectively  was estimated using the slope from linear regressions of the reaction monitored during minutes 3–10. Under these conditions, we were able to calculate the values of K0.5 3.9 ± 0.5 μM for NendoUhex, and Km of 7.4 ± 1.0 μM for NendoUmon, indicating that the hexamer form has an increased affinity for the substrate  RNA oligo, with or without Mn and using EDTA as control. Manganese was not observed to be crucial for the RNA cleavage, and EDTA does not seem to have any deleterious effect on the reaction  from an adjacent nsp15 chain Figure , but notet al. .The binding of dsRNA to the NendoU surface also revealed that the interactions seem to be mediated by the positively charged residues of NendoU and the phosphate groups of the ribonucleosides Figure , D. The hex in different pHs and buffers, with resolution ranging from 2.5 to 3.5 Å  and HEPES at pH 7.5 (a less active condition), detailed in During our cryo-EM studies, we were able to elucidate multiple high-quality models of NendoUto 3.5 Å . Based oet al. , we observed that the generated crystals were obtained in an orthorombic space group with 55% solvent content and biological units packed side by side as observed in the cryo-EM oligo(dT) samples, as exemplified here in PDB 7KF4 (mon and NendoUhex when incubated with other DNA sequences such as primers (data not shown), suggesting that this packing is somehow induced by the presence of DNA. If we compare the arrangement of adjacent hexamer units in the crystal structure  allowed us to depict the contacts taking part in the filament pattern, which involve the open form of the switch region of two adjacent hexamers, including the hydrogen bounds between Gln187 and Gly150, Asn136 and Thr114, Gly150 and Gnl187, and Gln152 and Gln188 Figure . FurtherTo identify starting points for new therapeutics, during the initial months of COVID-19, the XCHEM team and international collaborators performed large crystallographic fragment screens against multiple SARS-CoV-2 proteins, including the viral Main protease, the Nsp3 macrodomain and the helicase Nsp13 , which lP63, containing two NSP15 monomers in the asymmetric unit arranged in a dihedral symmetry, which can be symmetrically expanded to reveal the full hexameric form of NendoU inside the crystal lattice. Three libraries of compounds were used for soaking, i.e. the XCHEM Poised Library , so that we can map all the identified druggable cavities in the context of the hexameric complex Figure . Also, wUnfortunately, the NendoU active site, which would be a primary target for the development of a competitive inhibitor, was occupied by a citrate molecule that originated from the crystallization condition in all refined datasets Figure , which rThe second cluster (cluster 2) mapped is in a conserved region of the MD, which is a critical region of the oligomerization interface Figure . Here weAnother possible allosteric site for the development of new inhibitors is that identified in the third cluster (cluster 3), which is also a conserved region located at the bottom of the MD, where we found a cavity containing fragments Z2697514548, Z1424343998, Z425449682 and CSC000178569, forming different contacts with residues Arg135 and Lys191 Figure . The strThe most curious site identified in our experiments was cluster 4, which is a conserved site located in the interfaces of the ND and MD with the NSP15 domain of the neighboring monomer, in a cavity located inside the barrel shape of NendoU Figure . In addiBesides the four identified clusters, we were also able to map several different druggable cavities spread across the hexamer structure, including fragments EN300-1605072, Z2443429438, Z425449682, Z59181945, Z239136710, Z1530301542, Z18197050, EN300-100112, Z319891284, Z68299550, Z56900771 and Z31504642, which are all depicted in During our analysis of fragment screening data, we identified a set of datasets that had a completely disrupted chain A active site, causing an extreme increase in the B-factor of this region . A detaiAny other data can be supplied upon reasonable request. Crystallographic coordinates and structure factors for all structures have been deposited in the PDB with the following accession codes 5S6X, 5S6Y, 5S6Z, 5S70, 5S71, 5S72, 5SA4, 5SA5, 5SA6, 5SA7, 5SA8, 5SA9, 5SAA, 5SAB, 5SAC, 5SAD, 5SAE, 5SAF, 5SAG, 5SAH, 5SAI, 5SBF, 7N7R, 7N7U, 7N7W, 7N7Y, 7N83, 7KEG, 7KF4, 7KEH, 7RB0, 7RB2 and 7ME0. EMD data are available with the following accession codes EMD-24391, EMD-24392 and EMD-23786.gkad314_Supplemental_FilesClick here for additional data file.


Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni.The ShK toxin from T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity.We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni.Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of The online version contains supplementary material available at 10.1186/s12915-023-01617-y. In contKT) domains spanning multiple kingdoms documented in the Simple Modular Architecture Research Tool (SMART) database  in 0.1% formic acid [FA]) in solvent A (0.1% FA) over 18 min before a step gradient to 95% B for 18 min at a flow rate of 0.5 µL/min. The eluting peptides were analysed with an in-line Bruker timsTOF Pro operated in positive, data-dependent analysis, parallel accumulation-serial fragmentation (DDA-PASEF) mode with a cycle time of 0.5 s and an m/z range of 350–2200. The resulting MS and MS2 spectra were manually compared to a list of all possible peptic fragment precursors and their fragment ions estimated from all theoretical disulphide permutations. The disulphide bond connectivities identified in the recombinant peptide were confirmed with a sample of a natural peptide from . , where Y is reciprocal of the RCF and [toxin] is the molar concentration of SA8. The reciprocal of the slope yielded the IC50 value.Voltage protocols: For measurement of hKof KV1.2  200-ms-lDrosophila melanogaster was assessed using an injection activity assay [D. melanogaster used for this experiment ranged from 0.8 to 1.1 mg. Eight D. melanogaster were used as an injection control group and administered 50 nL of water. Five doses of SA8  were each administered to eight D. melanogaster , with observations of paralysis and mortality made at 2 and 24 h post-injection (Additional file The activity of the recombinant SA8 peptide in ty assay . The masAdditional file 1: Table S1. Repeats masked in the genomes of A. tenebrosa and T. stephensoni.Additional file 2: Table S2. CAFE was used to identify expanded gene families encoding putative toxinsacross actiniarian superfamilies Actinioidea, Edwardsioidea and Metridioidea. Species: Telmatactis = T. stephensoni; Paraphelliactis = P. xishaensis; Exaiptasia = E. diaphana; Actinia = A. tenebrosa; Nematostella = N. vectensis. Uncharacterised toxin gene families have been designated U# or Z#.Additional file 3: File S1. Supporting data values for figures. Fig. 2A_I. MCScanX duplication classification. Fig. 2A_II. MCScanX collinear blocks. Fig. 3A.A. tenebrosa SA8 expression matrix. Fig. 3B.T. stephensoni SA8 expression matrix. Fig. 4A.T. stephensoni WGCNA module membership. Fig. 4B.A. tenebrosa WGCNA module membership. Fig. 6. SA8 phylogenetic distance matrix. Fig. 8_Fig. S9. patch clamp electrophysiology measurements.Additional file 4: Figure S1. Sequence and structure of T. stephensoni SA8 sequences. Alignment of SA8 gene and peptide sequences from T. stephensoni, with conserved cysteine framework, glycine residue, and FA dyad highlighted. The nine clustered genes of T. stephensoni are composed of 3–5 microexons and large introns. The inverted T. stephensoni SA8 gene is indicated in red.Additional file 5: Figure S2. SDS-PAGE gel stained with Coomassie Blue showing samples of the His6-MBP-SA8 fusion protein obtained during different steps of expression and purification. Lane 1: molecular mass standards; Lanes 2 and 3: E. coli cells pre- and post-induction with IPTG; Lane 4: sucrose extract; Lane 5: periplasmic extract; Lanes 6 and 7: purified MBP fusion protein before and after cleavage with TEV protease.Additional file 6: Figure S3. RP-HPLC purification of recombinant SA8. The peptide was separated on a Vydac C18 columnusing a flow rate of 1 mL/min and a 40 min linear gradient of 30–50% solvent B, as indicated by the red line. LC-MS profile of oxidised SA8 after purification using RP-HPLC. The observed mass is consistent with formation of three disulfide bonds.Additional file 7: Figure S4. Comparison of LC-MS profiles for co-eluted native and recombinant SA8. LC-MS profile of the native SA8 peptide. LC-MS profile of the recombinant SA8 peptide.Additional file 8: Table S3. Direct mass spectrometric characterisation of disulfide linkages in the recombinant SA8 peptide. The main diagnostic pepsin fragments appeared as MS2 precursors with high intensity.Additional file 9: Figure S5. Full 1D 1H NMR spectrum of recombinant SA8 recorded on Bruker 600 MHz NMR spectrometer at pH 3.5 and 298 K and 256 scans. Expanded amide/aromatic region.Additional file 10: Figure S6. 1D 1H NMR spectra of SA8 recorded on Bruker 600 MHz NMR spectrometer. Spectra were acquired at pH 3.5 and different temperatures over the range 5–30 °C. Expanded amide/aromatic region.Additional file 11: Figure S7. 1D 1H NMR spectra of SA8 recorded on Bruker 600 MHz NMR spectrometer. Spectra were acquired at 298 K using 256 scans, over the pH range 2-7. Expanded amide/aromatic region.Additional file 12: Figure S8. 1D 1H NMR spectra of SA8 recorded on Bruker 600 MHz NMR spectrometer. Spectra were acquired at pH 3.5 and 298 K using 256 scans. Expanded amide/aromatic region. N.B. The sharp strong peaks in the spectra are from low molecular weight molecules.Additional file 13: Figure S9. SA8 has no effect on several voltage-gated and Ca2+-activated ion channels. Current traces recorded before application of SA8, after 1-2 min perfusion with SA8, and after perfusing the recording chamber with control solutions. Data are shown for the following channels: HkV1.1, hKV1.3, hKV10.1, hKV11.1, mKCa1.1, hNaV1.7, hKCa3.1, hTRPA1, and hTRPV1. For details on the expression systems, solutions, and voltage protocols, see Materials and Methods. For hKCa3.1, hTRPA1, and hTRPV1, the currents were recorded in response to a voltage ramp, corrected for ohmic leakage and then displayed as a function of test potential. The horizonal dashed line shows the zero current level and the vertical dashed line indicates the expected reversal potential for K+ .Additional file 14: File S6. Injection activity assay in Drosophila melanogaster.Additional file 15: Table S4. Accession numbers for tissue samples of A. tenebrosa and T. stephensoni.


Labeo rohita) exposed to graded concentrations of copper sulfate. For this purpose, L. rohita fish (n = 72), weighing ~200–215 g, were randomly divided into four experimental groups and then exposed to acute doses of CuSO4, i.e., control, 0.28, 0.42, and 0.56 μgL−1. For comparative analysis of hematological and biochemical changes, blood/serum samples were obtained on 12, 24, and 36 days. Overall, the body weight of fish decreased with the time and dose of CuSO4; as the dose increases, body weight decreases. Dose and time-dependent results were observed in other parameters also. Results showed a significant increase in leukocytes, whereas red blood cells count, Hb, and Hct were significantly reduced in treated groups compared to the control. The mean corpuscular hemoglobin (MHC) and mean corpuscular hemoglobin concentration (MCHC) showed a non-significant decrease in treated groups compared to the control group. Serum biochemical parameters, including total proteins, albumin, and globulin, decreased significantly (p < 0.05). At the same time, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glucose, and cholesterol were significantly (p < 0.05) increased in the treated groups compared to the control group. Significantly (p < 0.05) increased levels of lipid peroxidation while decreased values of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (RGSH) in the blood of fish were recorded. Histopathological examination of fish gills, liver, and kidneys showed inflammation and degenerative changes due to CuSO4 exposure. In the brain tissue, degenerative changes like neuron necrosis, intracellular edema, cytoplasmic vacuolization, and congestion were observed. In conclusion, the study indicates that exposure to copper sulfate, even in smaller concentrations, can cause adverse hematological and histopathological changes in L. rohita fish.Despite being an essential trace element for numerous metabolic processes and micronutrients, copper (Cu) has induced adverse effects on the environment and public health due to its continuous and widespread use for the last several decades. The current study assessed the hematological and histopathological alterations in the freshwater fish ( Increased agricultural activities and extensive industrialization ultimately enhance the levels of various metals in the aquatic environment. Copper (Cu), an essential micronutrient for humans and animals, is an important and vital trace element needed for the multiple normal metabolic functions in various living organisms , 2. Copp4) in various sectors like building materials, agricultural sector, electronic industry, water pipes, transportation sector, intra-uterine contraceptive devices, and as an antifungal agent and growth promoter in poultry has also induced toxicological impacts in the exposed species (4) negatively affected male Nile tilapia's reproductive function , were collected from a local fish hatchery and breeding center. All the fish were immediately transported to the laboratory and reared in glass aquaria having 100 L of water at 26 ± 1.0 °C and photo-regime (12/12 h) light/dark for 2 weeks under laboratory conditions. Before the start and during the trial, all the fish were offered Optimum Aquarium Fish Food having 28% crude proteins . The feed was provided to all the fish at 2–3% of body mass twice daily. After 2 weeks, the fish were divided randomly into equal four groups (A-D) having 18 fish in each group. Copper sulfate (CuSO4) of analytical grade was procured from the local market. The concentrations of CuSO4 were selected following the previous studies   . A blood−1) . Total p−1) . After c−1) .Randomly selected, six fish were weighed on the 12th, 24th, and 36th day of the trial, then an autopsy was conducted. Visceral organs like the kidneys, brain, gills, and liver were dissected and weighed immediately. The relative weight was measured as a percent of the body weight. Similarly, the liver, kidneys, brain, and gills were preserved in 10% paraformaldehyde for microscopic studies. After fixation, all the visceral tissues were processed for histopathological changes using eosin and hematoxylin staining procedures . Tissue TBARS values were determined according to the procedure already described , while rpost hoc Tukey's test to determine the significant variation at p < 0.05.The data thus collected from hemato-biochemical, oxidative stress, and antioxidant enzymic parameters were presented as means ± standard error (SE). The data were subjected to ANOVA  using statistical software  followed by 4  during the trial. Clinical and behavioral signs include loss of equilibrium, mucus secretion from mouth and gills, air gulping, increased swimming, rapid operculum movement, bulging eyes, loss of coordination, erratic swimming, and swimming in isolation detected in the treated fish. The severity of various clinical and behavioral signs increases parallelly with increased concentration and exposure time to CuSO4 .p < 0.05) in group D (CuSO4: 0.56 μgL−1) compared to the control group on the 36th experimental day  than the control group on experimental days 12, 24, and 36 (p < 0.05) in the control group at CuSO4 dose 0.48 μgL−1 on experimental day 36. The absolute weight of gills increased significantly (p < 0.05) at CuSO4 dose 0.56 μgL−1 than the control group on the 36th experimental day  at CuSO4 doses of 0.48 μgL−1 and 0.56 μgL−1 on experimental day 36th than the control group (p < 0.05) on experimental days 24th and 36th in groups C (CuSO4: 0.48 μgL−1) and D (CuSO4: 0.56 μgL−1) compared to the control group. As far as the relative weight of the kidneys is concerned, that showed a significant (p < 0.05) increase on experimental days 12th, 24th, and 36th in group D (CuSO4: 0.56 μgL−1) and also in group C (CuSO4: 0.48 μgL−1) on experimental day 24 compared to control group (p < 0.05) relative weight of the brain increased on experimental days 12th, 24th, and 36th in groups C (CuSO4: 0.48 μgL−1) and D (CuSO4: 0.56 μgL−1) compared to the control group (Results showed that the relative liver weight of fish increased significantly (ol group . In contol group . Signifiol group .4 are presented in p < 0.05) while WBC counts and neutrophils increased significantly (p < 0.05) on experimental days 12, 24, and 36 in high dose group (CuSO4: 0.56 μg.L−1) compared to control group. In addition, hemoglobin and hematocrit decreased significantly (p < 0.05) at CuSO4 dose 0.42 μg.L−1 on experimental day 36 compared to the control group. WBCs counts also increased significantly (p < 0.05) on experimental day 24 and 36 at CuSO4 dose 0.42 μg.L−1 compared to control group (p > 0.05) throughout the experiment as compared to the control group.Different hematological parameters of fish exposed to several concentrations of CuSOol group . MHC and4 are presented in p < 0.05) in the blood serum of fish treated with CuSO4 dose 0.56 μg.L−1 on experimental days 24 and 36 and also with CuSO4 dose 0.42 μg.L−1 on experimental day 36 compared to control group. In contrast, ALP and ALT increased significantly (p < 0.05) in fish treated with CuSO4 dose 0.56 μg.L−1 on experimental days 12, 24, and 36 and also with CuSO4 dose 0.42 μg.L−1 on experimental day 36 compared to control group. Total proteins and albumin concentration decreased significantly (p < 0.05) with the treatment of CuSO4 dose 0.56 μg.L−1 on experimental days 24 and 36, and total proteins also decreased significantly (p < 0.05) with the treatment of CuSO4 dose 0.42 μg.L−1 on experimental day 36  increased quantity of TBARS in fish treated with CuSO4 dose 0.56 μg.L−1 on experimental days 24 and 36 and with CuSO4 dose 0.42 μg.L−1 on experimental day 36 compared to the control group. The TBARS also increased significantly (p < 0.05) with the treatment of CuSO4 dose 0.42 μg.L−1 on experimental day 36 (p < 0.05) decreased concentrations of different antioxidant enzymes, including reduced glutathione, superoxide dismutase, and catalase in erythrocytes of fish treated with CuSO4 dose 0.56 μg.L−1 on experimental days 24 and 36 and also with CuSO4 dose 0.42 μg.L−1 on experimental day 36 compared to control group (The results of oxidative stress (Thio-barbituric acid reactive substances) and different antioxidant enzymes  are presented in l day 36 . There wol group .4 dose 0.28 μg.L−1 and experimental day 12 and increased significantly (p < 0.05) with increasing the dose of CuSO4 from 0.42 μg.L−1 to 0.56 μg.L−1 and experimental day from 24 to 36. The degree of histopathological lesions was moderate to severe  fish has been reported with exposure to low (10 BBP) compared with high (100 BBP) dose of CuSO4 (4 showed a significant (p < 0.05) reduction in the treated groups compared to the control group. Similarly, it has been reported that Epinephelus coioides supplemented with CuSO4 and copper nanoparticles (Cu-NPs) rearing water exhibited lower weight gain than the control group (The current study showed that various concentrations of CuSOof CuSO4 . In the ol group . A decreol group .6/mm3), hematocrit (34.14 ± 2.39 vs. 24.70 ± 0.58 %), and hemoglobin (8.96 ± 0.23 vs. 6.91 ± 0.18 g.dL−1), and lymphocytes (18.61 ± 0.54 vs. 14.85 ± 0.16 %) were decreased significantly (p < 0.05) after the exposure to CuSO4 (0.28 μg.L−1) on all experimental days. Similarly, CuSO4 (0.05 mg/L) exposure to fish Oreochromis niloticus had been reported to significantly (p < 0.05) decrease erythrocytes (1.78 ± 0.03 vs. 1.41 ± 0.02 106/μL), hematocrit (31.95 ± 0.74 vs. 24.83 ± 0.37 %), and hemoglobin (8.38 ± 0.09 vs. 5.94 ± 0.12 g/dL) values  values . It is a4 itself . The low4 itself , 53, 56.4 itself , yellow 4 itself , and Cat−1), all biochemical parameters including serum urea (8.04 ± 0.05 vs.11.57 ± 0.05 mg.L−1), creatinine (1.28 ± 0.02 vs. 1.94 ± 0.02 mg.L−1), cholesterol (158.9 ± 1.50 vs. 188.3 ± 3.50 mg.L−1), glucose (30.7 ± 1.19 vs. 37.4 ± 1.19 mg.L−1), triglycerides (156.3 ± 3.41 vs.183.7 ± 2.41 mg.L−1) along with serum enzymes i.e., ALT (23.10 ± 0.13 vs. 32.60 ± 0.13 UL−1), AST (13.80 ± 0.13 vs.18.10 ± 0.13 UL−1), ALP (24.40 ± 0.13 vs. 33.90 ± 0.13 UL−1) and LDH (253.3 ± 2.61 vs. 274.4 ± 5.61 UL−1) increased significantly (p < 0.05) in L. rohita with the exposure of CuSO4 (p < 0.05) in Nile tilapia (Oreochromis niloticus) with the treatment of both CuSO4 (15 mg/L) and CuO-NP  whereas RGSH (0.13 ± 0.02 vs. 0.06 ± 0.02 μmol GSH/mg protein), SOD (2.38 ± 0.15 vs. 1.39 ± 0.11 units/mg protein) and CAT (2.46 ± 0.12 vs. 1.37 ± 0.22 units/mg protein) decreased significantly (p < 0.05) in red blood cells of L. rohita with the exposure of CuSO4 (p < 0.05) in Nile tilapia (Oreochromis niloticus) with the treatment of both CuSO4 (15 mg/L) and CuO-NP  increased significantly  injury/damage leading to disfunction e stress . In hepae stress . Direct e stress , 72, 87 exposure . Injury exposure . Copper exposure –91 whichSimilarly, Abdel-Latif et al.  conducteThe gills are the first organ affected by exposure to CuSO4 in water . In the 4 dose and time-dependent histopathological alterations in brain tissues including congestion, cytoplasmic vacuolization, intracellular edema, and neuron necrosis were recorded. In the published literature, CuSO4 or CuO-NPs post disclosure, neuropil degeneration was obvious in Nile tilapia exposed to copper  might result in physiological and histopathological alterations in multiple fish tissues. Based on the results derived from this study, further studies are proposed to find out the more suitable dose of CuSO4 for regular use to decontaminate fish ponds. It is also suggested that due to possible effects on the ecosystem, aquatic organisms, and animal and human health, we should regularly monitor exposure to CuSO4 in waters and continuously observe the environment/ecosystem. Hence, we must prioritize studies about environmental monitoring of pollutants and their possible effects on different organisms.Many changes in the hematological and serum biochemical parameters and histopathological status in While studying copper sulfate toxicity in fish in the past, people have reported mostly hematological or biochemical parameters singly or in combination. Whereas, in this study, we have reported comprehensively and simultaneously, physical, hematological, or biochemical, oxidative stress parameters along with gross and histopathology of fish exposed to various doses and at various time intervals.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Bioethics Committee, The Islamia University Bahawalpur, Pakistan.SN and RH had the initial idea for the study. They were involved in the experiment, laboratory, data analysis, writing and development of the article, and the final proof stage. RH, SJ, and AC were involved in experimental studies, laboratory studies, and the article's writing. ML was involved in experimental studies, laboratory studies, and data analysis. ZG and AK performed and interpreted the statistical analysis. ZR arranged the facilities . AK valuable suggestions regarding histopathological studies. All authors have read and approved the final version of the manuscript.


Dictyophora indusiata is well-known for its morphological elegance, distinctive taste, high nutritional value, and therapeutic properties. In this study, eighteen compounds (1–18) were isolated and identified from the ethanolic extract of D. indusiata; four (1–4) were previously undescribed. Their molecular structures and absolute configurations were determined via a comprehensive analysis of spectroscopic data . Seven isolated compounds were examined for their anti-inflammatory activities using an in vitro model of lipopolysaccharide (LPS)-simulated BV-2 microglial cells. Compound 3 displayed the strongest inhibitory effect on tumor necrosis factor-α (TNF-α) expression, with an IC50 value of 11.9 μM. Compound 16 exhibited the highest inhibitory activity on interleukin-6 (IL-6) production, with an IC50 value of 13.53 μM. Compound 17 showed the most potent anti-inflammatory capacity by inhibiting the LPS-induced generation of nitric oxide (NO) (IC50: 10.86 μM) and interleukin-1β (IL-1β) (IC50: 23.9 μM) and by significantly suppressing induced nitric oxide synthase (iNOS) and phosphorylated nuclear factor-kappa B inhibitor-α (p-IκB-α) expression at concentrations of 5 μM and 20 μM, respectively (p < 0.01). The modes of interactions between the isolated compounds and the target inflammation-related proteins were investigated in a preliminary molecular docking study. These results provided insight into the chemodiversity and potential anti-inflammatory activities of metabolites with small molecular weights in the mushroom D. indusiata.As an edible and medicinal fungus,  Dictyophora indusiata, belonging to the family Phallaceae (phylum Basidiomycetes) of the Agaricomycetes class of fungi, is famous for its beautiful morphological features. Due to its delicious taste, distinctive fragrance, and medicinal properties, D. indusiata has a long history as a healthy food and as a component of folk medicine  oxazine-6-methylal (11) + . 1H NMR data  and 13C-NMR data  are shown in Compound 15H22O2, M = 234.32, orthorhombic, a = 7.4315(7) Å, b = 13.1582(12) Å, c = 26.367(2) Å, U = 2578.3(4) Å3, T = 111.6(9), space group C2221 (no. 20), Z = 8, μ(Cu Kα) = 0.612, 4187 reflections measured, 2412 unique (Rint = 0.0675) which were used in all calculations. The final wR(F2) was 0.1293.Crystal data: C2: yellowish oil, UV(MeOH) λmax 235.7 nm; HRESIMS m/z: 235.1694 [M + H]+ . 1H NMR data  and 13C-NMR data  are shown in Compound 3: brown crystal, UV(MeOH) λmax 229.8 nm; HRESIMS m/z: 306.0981 [M + H]− . 1H NMR data  and 13C-NMR data  are shown in Compound 15H15NO7, M = 321.28, trigonal, a = 26.3585(15) Å, c = 5.4846(4) Å, U = 3300.1(4) Å3, T = 113(2), space group R3 (no. 146), Z = 9, μ(Cu Kα) = 0.998, 5982 reflections measured, 2699 unique (Rint = 0.0302), which were used in all calculations. The final wR(F2) was 0.1274.Crystal data: C4: yellowish oil, UV(MeOH) λmax 277.1 nm; HRESIMS m/z: 225.1482 [M + H]− . 1H NMR data  and 13C-NMR data  are shown in Compound 1, 3, 4, 11, 15, 16, and 17 were selected for in vitro investigations of anti-inflammatory activities using an LPS-stimulated BV-2 microglial cell model. Three groups were designed for the experiments: the normal cell group, where the cells were cultured under normal growth conditions; the LPS group, where the cells were treated with LPS but without the tested compounds; and the sample group, where the cells were treated with LPS plus the tested compounds.Compounds 3 cells/well and cultured overnight in an incubator at 37 °C with 5% CO2. Then, the cells were treated with different concentrations of the selected compounds diluted with DMEM. After 2 h, LPS (1 μg/mL) was added. After 24 h of incubation, 20 μM MTT was added to each well and the cells were incubated at 37 °C for 4 h. Afterward, the supernatant was discarded, and 150 μL of DMSO was added to each well to dissolve the formazan. The absorbance  of each well was measured at 490 nm using a multilabel plate reader . The cell viability results were calculated as percentages.The cytotoxic effects of the selected compounds on the BV-2 cells were evaluated using an MTT assay . Briefly3 cells/well, cultured overnight in an incubator at 37 °C with 5% CO2, and then cultured with LPS (1 μg/mL) in the absence or presence of the tested compounds for 24 h. The culture supernatants were collected for the measurements. The total nitric oxide level was measured using the NO assay kit. The TNF-α, IL-1β, and IL-6 concentrations were quantified using mouse ELISA kits. All operations were conducted according to the manufacturer’s instructions.The tested compounds were diluted in a gradient with DMEM. The BV-2 cells were seeded into 96-well plates at a density of 8 × 10p-IκB-α were measured using a Western blot analysis [5 cells/well. Then, the cells were washed twice with 4 °C phosphate-buffered saline (PBS) and harvested. Next, 200 μL of radioimmunoprecipitation assay (RIPA) buffer was added to each well, and the lysate was centrifuged at 12,000× g for 20 min after incubation at 4 °C for 30 min. The protein concentration was determined using a bicinchoninic acid (BCA) assay. The protein was collected for Western blot analyses of iNOS and p-IκB-α. After being resolved using SDS/PAGE loading buffer, the protein was electrophoretically separated at 120 V for 90 min and transferred to polyvinylidence difluoride (PVD) membranes at 300 mA for 85 min. Then, the PVD membranes were blocked with 5% skim milk in phosphate-buffered saline containing 0.05% Tween-20 (PBST) for 120 min at room temperature. The cells were incubated overnight at 4 °C in diluted primary antibodies (anti-iNOS and anti-p-IκB-α). The membrane was washed with PBST three times and incubated in a (horseradish peroxidase) HRP-conjugated secondary antibody solution for 120 min at room temperature. Finally, an appropriate amount of enhanced chemiluminescence reagent was added to detect the protein. The data for iNOS and p-IκB-α were normalized on the basis of GAPDH levels.The protein expressions of iNOS and analysis . The teshttp://www.rcsb.org/, accessed on 1 February 2023). The structure of the IκB-α/NF-κB complex (PDB ID: 1IKN) was modeled using SwissModel  to remove the repeats of subunits from the heterodimer [3, 15, 16, and 17 were created and optimized to lower-energy conformers with the Maestro Build Panel, and they were used as the ligands for the molecular docking simulations. The glide ligand docking and induced-fit docking modules in Maestro 11.9 software were employed for the docking study and for the analysis of the results [https://www.rbvi.ucsf.edu/chimerax, accessed on 1 February 2023).The protein structures of TNF-α (PDB ID: 7KP8), IL-1β (PDB ID: 8I1B), iNOS (PDB ID: 4UX6), and IL-6 (PDB ID: 2L3Y) were downloaded from the Protein Data Bank . A p-value < 0.05 was considered statistically significant.Each experiment was repeated three times, with three replicates within each experiment. The data are expressed as mean ± standard deviations (SD). IBM SPSS software  was used to generate graphs and perform statistical analyses. Data were analyzed using a Student’s D. indusiata ethanolic extract resulted in the isolation and identification of 18 compounds (1–18), including three previously undescribed sesquiterpenoids  and one previously undescribed quinolone derivate (3). Their structures and absolute configurations were elucidated using spectroscopic methods and chemical analyses. Among them, compound 1 was the first natural product identified from fungi with an unusual molecular skeleton of antsorane. Seven isolated compounds were evaluated for anti-inflammatory activities in an LPS-stimulated BV-2 microglial cell model. Compound 3 had the best inhibitory effect on TNF-α secretion. Compound 16, an ergosterol derivative with a rearranged tetracyclic skeleton, showed the most potent activity on the suppression of IL-6 generation. The 5α,6α-epoxy-7-sitosterol compound 17 exhibited an anti-inflammatory effect by inhibiting the production of NO and IL-1β and the expressions of iNOS and p-IκB-α. The molecular docking study conducted in this research indicated that the tested compounds could exert their anti-inflammatory activities by binding to the hydrophobic pocket of the corresponding protein. These findings suggest that, as D. indusiata is an edible and medicinal mushroom, its anti-inflammatory property would be at least partially contributed by the presence of a series of compounds with small molecular weights as active constituents. The results of this study demonstrate the chemodiversity and biological potential of the small-molecule metabolites in D. indusiata. They provide a foundation for future investigations into the molecular basis of the pharmacological effects of the mushroom Dictyophora indusiata.A chemical investigation of the ethyl acetate fraction of


In this study, suppression by siRNA-mediated knockdown and pharmacological sensitivity of VGCC currents evidenced molecular and functional expression of N-type Cav2.2 and T-type Cav3.1 in AVP neurons under normotonic conditions. Also, both the Cav2.2 and Cav3.1 currents were found to be sensitive to flufenamic acid (FFA). TTX-insensitive spontaneous action potentials were suppressed by FFA and T-type VGCC blocker Ni2+. However, Cav2.2-selective ω-conotoxin GVIA failed to suppress the firing activity. Taken together, it is concluded that Cav2.2 and Cav3.1 are molecularly and functionally expressed and both are sensitive to FFA in unstimulated rat AVP neurons. Also, it is suggested that Cav3.1 is primarily involved in their action potential generation.Arginine vasopressin (AVP) neurons play essential roles in sensing the change in systemic osmolarity and regulating AVP release from their neuronal terminals to maintain the plasma osmolarity. AVP exocytosis depends on the Ca Howechannels . Thus, f. 1999) . On the asically . Expresse to AVP  and PACAe to AVP . Direct nditions . However999 . On channels , 15. Theectively . Express neurons . In cont2+ channels are activated at lower voltages than high-threshold L-, P/Q-, N- and R-type Ca2+ channels. Under physiological recording conditions, the apparent activation threshold for T-type Ca2+ channels is − 50 to − 70 mV . Hocreening , 36. In 2+ channels in AVP neurons are sensitive to FFA (Fig. −1, or 21 to 71 μM, within 1.5 h [The present study also, for the first time, demonstrated that both N-type Cav2.2 and T-type Cav3.1 CaFFA Fig. . FFA hasin 1.5 h . Since Fin 1.5 h .+ and Ca2+ conductances [+ channels and of FFA- and Ni2+-sensitive T-type Cav3.1 Ca2+ channels (Fig. 2+ channel activity from inactivation, in light of previous data of around 90% recovery from short inactivation at 400 ms after firing of neuronal T-type Ca2+ channels [2+ and ω-CgTx (Fig. Somatodendritic action potentials of rat supraoptic MCNs were previously shown to arise from co-activation of Naels Fig. . The timgTx Fig. b, c. SinIn dissociated rat AVP neurons under normotonic conditions, N-type Cav2.2 and T-type Cav3.1 VGCCs were found to be expressed and predominantly functioning, and be sensitive to FFA. Also, it is suggested that T-type Cav3.1 VGCC is primarily involved in their action potential generation in AVP neurons under normotonic conditions.


Objective: To investigate the efficacy and safety of chemotherapy in treating Ph+ ALL based on flumatinib.Methods: The clinical data of 29 patients with Ph+ ALL receiving flumatinib-based chemotherapy in Sichuan Provincial People’s Hospital from January 2020 to January 2023 were collected for analysis, with the concentrations of TKI in the peripheral blood, bone marrow, and cerebrospinal fluid of some patients monitored, Cytological experiments on SUP-B15 were conducted in a Ph+ ALL cell line.Results: A total of 29 patients were enrolled, showing the induced CR, 3-month CR, and 6-month CR rates of 96.3%, 87.5%, and 86.7%, respectively after flumatinib-based chemotherapy. The negative conversion ratio of MRD was 82.6%, 91.3%, and 95.6% in 1, 2, and 3 months after treatment, respectively, with 4.3% of patients failing the conversion in 3 months after treatment. The rates of MMR were 73.9%, 87.5%, and 93.3% in 1, 3, and 6 months after treatment, and CMR of 52.2%, 62.5%, and 73.3%, respectively. Among the 29 patients, 11 (37.9%) received transplant and the continuous flumatinib for 1 year after transplantation. The deep remission was maintained in all patients up to the time of follow-up, with the median follow-up of 12 months (1–33 months), progression-free survival (PFS) of 11 months (1–33 months), and median overall survival (OS) of 12 months (1–33 months). The adverse reactions mainly referred to myelosuppression, liver insufficiency and infection that were generally tolerable. In terms of blood concentration, the concentration of flumatinib was ordered as bone marrow > serum > cerebrospinal fluid in Ph+ ALL bone marrow. In contrast, the concentration of dasatinib and imatinib was ordered as serum > bone marrow > cerebrospinal fluid. At the same time, flumatinib has a high probability to cross the blood-brain barrier, while the concentration of cerebrospinal fluid in the patients using Dasatinib was lower compared to the lower limit of detection in this study. Compared with Imatinib and Dasatinib, flumatinib exerted the most potent inhibitory effect on Ph+ ALL cell lines according to pharmacodynamic analysis of SUP-B15 cells.Conclusion: Flumatinib combined with chemotherapy could achieve good efficacy and safety in treating Ph+ ALL, with flumatinib in a high probability of crossing the blood-brain barrier. Flumatinib could be a superior choice to Dasatinib and Imatinib in cell experiments. Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a common type of acute lymphoblastic leukemia (ALL), with the banded analysis of t  , accounting for about a quarter of ALL adults , typicalClinical data of 29 patients with Ph+ ALL receiving flumatinib treatment in Sichuan Provincial People’s Hospital from January 2020 to January 2023 were collected for the retrospective analysis, all of whom had passed ethical review.① Diagnosed as Ph+ ALL according to the NCCN guidelines; ② Age ≥ 18 years old; ③ Sign informed consent; ④ Administrated with flumatinib for at least 1 month.① Age < 18 years old; ② Pregnant, preparing for pregnancy or lactating women; ③ Patients with secondary myelodysplastic syndrome (MDS), chronic myeloproliferative disease (MPN) and lymphoproliferative disease. ④ CML-LBC. ⑤ Malignant tumors with other progression; ⑥ Serious heart, liver, kidney organ insufficiency.Baseline data of blood routine, blood biochemistry, primitive + infantile lymphocyte count in newly diagnosed bone marrow cytology, flow cytometry, fusion gene, BCR-ABL fusion gene quantification, all-related gene mutation, chromosome, ABL kinase region mutation site, and hepatitis B and C liver screening were recorded. The followed-ups were carried out for some of the above indexes throughout the treatment.(1) tyrosine kinase inhibitors: Patients are instructed to use tyrosine kinase inhibitors as soon as possible after diagnosed.(2) Combination chemotherapy: all patients were treated with VP (Vincristine/Vindesine + prednisone) induction regimen in consolidation therapy, where 15 received Hyper-CVAD B (Methotrexate + Cytarabine + methylprednisolone) for consolidation therapy, and 5 for Hyper-CVAD A (cyclophosphamide + vincristine + doxorubicin/pirarubicin + dexamethasone), 2 for CAM (cyclophosphamide + cytarabine + 6-mercaptopurine), 1 (DDLD) for (dexamethasone + Pegaspargase + Nordoxorubicin + vindesine), and 1 for VP (Vinchnestine/vindesine + prednisone), two patients underwent induction therapy without consolidation, and three patients were lost during follow-up. Some patients were brided with blinatumomab, followed by allogeneic hematopoietic stem cell transplantation .(3) Three intrathecal injections of methotrexate, cytarabine, and dexamethasone were performed for at least six times during treatment to prevent central nervous system leukemia.The treatment process is shown in The efficacy of ALL was evaluated based on the criteria of the National Comprehensive Cancer Network (NCCN), primarily for complete response rate (CR) and molecular response (MMR and CMR). Here, MRD levels in bone marrow and cerebrospinal fluid were detected using multi-parameter flow cytometry, with the second generation gene sequencing and the decline of BCR-ABL gene monitored.The follow-up including outpatient, inpatient was carried out by telephone until 1 January 2023, with the median follow-up time of 12 months (1–33 months). Overall survival (OS) was defined as the time between initiation of flumatinib and the last follow-up or death, progression-free survival (PFS) time as the time from the start of flumatinib treatment until a change in the patient’s disease, such as disease progression or death.Adverse reactions were evaluated using the National Cancer Institute (NCI) Standard for Evaluating Common Adverse Reaction Terms Version 5.0 (CTCAE V5.0).The trough concentration of TKI was detected in 1, 2, and 3 months after treatment. Samples of all patients were collected 30 min ahead of medication on the same day, with 2 mL of peripheral blood, bone marrow, and cerebrospinal fluid samples collected for each patient. Flumatinib blood-brain barrier permeability = concentration of flumatinib in cerebrospinal fluid/concentration of serum samples collected simultaneously.Logarithmic growth SUP-B15 cells were inoculated in a 96-well plate, with the concentration set at 6 × 105/L. The concentrations of the flumatinib were set as 25, 12.5, 6.25, 3.12, 1.56, and 0.78 μmol/L, respectively, 160, 80, 40, 20, 10, and 5 μmol/L for dasatinib, respectively, 40, 20, 10, 5, 2.5, and 1.25 μmol/L for imatinib, respectively. Wells of zero setting and blank control were set, with 3 multiple wells in each group and incubated in 37°C, 5% CO2 incubator for 24 h. CCK-8 solution of 20 μL was added to each well and incubated for 4 h. An enzyme-labeled meter was employed to detect the absorbance.SUP-B15 cells of logarithmic growth stage were inoculated in a 24-well plate, with the concentrations of 1.56, 0.78, 0.39 μmol/L for flumatinib, 10, 5, 2.5 μmol/L for dasatinib, and 20, 10, 5 μmol/L for imatinib, respectively. Three multiple wells were set and incubated in a 5% CO2 incubator at 37°C for 24 h. After incubation, the cell suspension was collected for centrifugation, then the supernatant was discarded. The smear was re-suspended with 100 μL PBS, fixed with methanol for 10 min, rinsed with PBS and dried, stained with Garrey-Giemsa at working solution for 30 min, rinsed with UP water and dried. Cell morphology was observed and photographed using an inverted microscope.SUP-B15 cells of logarithmic growth stage were inoculated in 24-well plate, with the cell density adjusted to 1 × 106/L, the concentrations of flumatinib were set as 1.56 and 0.78 μmol/L, 10 and 5 μmol/L for dasatinib, and 20 and 10 μmol/L for imatinib, respectively. Three multiple wells were set up in both groups, then incubated at 37°C in 5% CO2 incubator for 24 h. The supernatant was removed after centrifugation at 4°C, washed twice with PBS, removed after centrifugation, and stained with AnnexinV-EGFP/PI double labeling. The outcomes were determined by flow cytometry within 1 h.SUP-B15 cells of logarithmic growth stage were inoculated into 24-well plates, with the cell density adjusted to 1 × 106/L. The concentrations of flumatinib were 3.12 and 1.56 μmol/L, respectively. Cells were collected, washed with PBS once, fixed after pre-cooled with 70% ethanol overnight, centrifuged to remove supernatant, washed with pre-cooled PBS, centrifuged to remove supernatant, added with a staining solution containing RNase A and PI, and detected by flow cytometry. Flow analysis was carried out on FlowJo software.p < 0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism version 9.4. The data were expressed as mean ± standard deviation or median. The Fisher exact probability test was performed when the total sample size was below to 40 or the minimum theoretical frequency below to 1. OS was evaluated by the Kaplan-Meier curve. The survival between groups was compared by log-rank test. Test level = 0.05, 9/L) were diagnosed for the first time. There obtained 19 patients (65.5%) with IKZF1 deletion mutation, 1 patient (3.4%) with RUNX1 gene mutation, 7 patients (24.1%) with negative gene mutations, and 3 patients (10.3%) refused gene testing. There categorized 22 (75.9%) P190-positive patients and 7 (24.1%) P210-positive patients in the BCR-ABL fusion genotype. There were 2 patients (6.9%) with HOX11 and 2 patients (6.9%) with WT1. According to genetic characteristics, 7 patients (27.6%) were normal in chromosome karyotype, 11 patients (37.9%) with Ph-positive alone, 6 patients (20.7%) with Ph-positive combined with other chromosome abnormalities, 1 patient (3.4%) with poor cell growth resulting in the failure to analyze the mitotic phase after multiple films, and 3 patients (10.3%) refused karyotype examination. Three patients (10.3%) had extramedullary infiltration, two (6.9%) with superficial lymph node enlargement, and one (3.4%) with arm mass.A total of 29 patients with Ph+ ALL were enrolled in this study, with a median age of 47 (18–85) years, including 17 males (58.6%) and 12 females (41.4%), with 24 high-risk patients (82.8%). A total of 16 cases (55.1%) with hyperleukocytosis (white blood cell count >30*10Twenty-one patients (72.4%) received flumatinib after the first diagnosis, and 8 patients (27.6%) turned to flumatinib from other tyrosine kinase inhibitors. Among the 8 patients with drug change, 2 patients (25.0%) chose to change due to abnormal liver function, 2 patients (25.0%) due to renal insufficiency, and 1 patient (12.5%) due to liver and kidney insufficiency, 2 patients (25.0%) due to pleural effusion, and 1 patient (12.5%) due to drug resistance and mutation in the ABL kinase region.After the administration of flumatinib combined with VP induction, the induced CR, 3-month CR, and 6-month CR rates achieved 96.3%, 87.5%, and 86.7%, respectively. The negative conversion rate of MRD was 82.6%, 91.3%, and 95.6% in 1, 2, and 3 months after treatment, respectively, with 4.3% of patients showing no negative conversion. The rates of MMR and CMR were 73.9%, 87.5%, and 93.3%, and 52.2%, 62.5%, and 73.3% in 1, 3, and 6 months after treatment, respectively. Among the 29 patients, 11 (37.9%) were transplanted and continued to use flumatinib for 1 year after transplantation. The deep remission was maintained in all patients up to the time of follow-up. Median follow-up was 12 months (1–33 months), with progression-free survival (PFS) of 11 months (1–33 months), and median overall survival (OS) of 12 months (1–33 months) .p > 0.05) (p = 0.074), suggesting no impact on long-term survival after switched to flumatinib  in first-line patients newly diagnosed and those switched from other TKI to flumatinib were provided in umatinib .p = 0.0372)  with the worse prognoses ( 0.0372) .p = 0.0211)  exhibited IKZF1 deletion mutation, where 17 could receive follow-up, and 2 were lost. Ten patients exhibited no IKZF1 (34.5%), where 9 could receive follow-up, and 1 was lost. Survival analysis was performed using the Kaplan-Meier method in both groups, which revealed the worse prognoses in patients with IKZF1 ( 0.0211) .p = 0.0386)  .p = 0.0143)  had MMR, where 2 exhibited no MMR in 3 months after treatment. The study population was divided into groups according to whether they had achieved MMR 3 months after treatment to compare OS status. The results indicated the prolonged OS of patients by the implementation of MMR in 3 months after treatment ( 0.0143) .p = 0.0066)  underwent autoHCT after consolidation therapy, all maintained deep remission. However, some old patients did not choose to enter autoHCT considering the high risk, economic reasons, incompatibility and other reasons. A total of 20 patients were classified into the high-risk group with consolidation therapy, of which 8 were transplanted immediately. The high-risk group was divided into two groups according to whether chose transplant, to compare the OS of patients. The results showed the significantly prolonged OS by autoHCT ( 0.0066) .p > 0.05).Survival analysis on the effects of sex, risk stratification, hyperleukocytosis at first diagnosis, different types of BCR-ABL, negative turn of MRD 1 month after treatment, and CMR in 3 months after treatment on OS was conducted, revealing no statistical difference  of grade II myelosuppression, 2 (8.3%) of grade III myelosuppression, and 18 (75.0%) of grade IV myelosuppression. The median minimum values for neutropenia were 0.15 (0–1.79) ×10^9/L, 56 (0–59) ×10^9/L for hemoglobin, and 21 (0–51) ×10^9/L for platelets. Most of the non-hematological adverse reactions were liver insufficiency, of which 6 patients (50.0%) had grade I liver function impairment, 3 patients (25.0%) with grade II liver function impairment, and 3 patients (25.0%) with grade III liver function impairment. The liver function of the above patients returned to normal after receiving symptomatic treatment, such as the stabilized liver cell membrane.After 1 month of flumatinib in Ph+ ALL patients, the concentrations of flumatinib in serum, bone marrow, and cerebrospinal fluid reached 32.5 ± 24.4 ng/mL (12–94 ng/mL), 128.9 ± 81.2 ng/mL (41.3–318 ng/mL) and 0.67 ± 0.61 ng/mL (0–1.5 ng/mL), respectively. The blood-brain barrier permeability of flumatinib was 3.01% ± 3.55% (0–8.48%). Two months after flumatinib, the concentration of flumatinib in serum and bone marrow reached 21 ng/mL and 35.4 ng/mL, respectively. After 3 months, the concentrations in serum, bone marrow, and cerebrospinal fluid were 46.22 ± 20.93 ng/mL (19–77 ng/mL), 148.9 ± 100.05 ng/mL (62.5–290 ng/mL), and 0.57 ± 0.43 ng/mL (0.3–1.2 ng/mL), respectively. The blood-brain barrier permeability of flumatinib was calculated as 1.39% ± 0.87% (0.39%–2.14%), with the concentrations of flumatinib in serum, bone marrow, and cerebrospinal fluid of 34.37 ± 14.53 ng/mL (25–51.1 ng/mL) and 119 ± 90.51 ng/mL (55–183 ng/mL), respectively, and 1.5 ng/mL, respectively. The blood-brain barrier penetration rate of flumatinib was calculated as 6%. Flumatinib concentrations in serum, bone marrow, and cerebrospinal fluid were 80.4 ng/mL, 203 ng/mL, and 0.9 ng/mL, respectively after 5 months, with the blood-brain barrier permeability as 1.12%. After 10 months, the concentrations in serum, bone marrow, and cerebrospinal fluid reached 38, 214, and 0.36 ng/mL, respectively, with the blood-brain barrier permeability of flumatinib as 0.95%. After 1 year, the concentrations in serum, bone marrow, and cerebrospinal fluid were 29.93 ± 9.09 ng/mL (19.8–41.9 ng/mL), 59.75 ± 21.71 ng/mL (44.4–75.1 ng/mL), and 0.9 ± 0.17 ng/mL (0.7–1 ng/mL), with the blood-brain barrier permeability as 2.86% ± 1.03% (1.67%–3.45%) .The concentrations of dasatinib in 3 Ph+ ALL patients and imatinib in 2 Ph+ ALL patients were also determined, the cerebrospinal fluid was <1 ng/mL in all. Distinguished from the patients using flumatinib, the concentration of drugs in those using dasatinib was higher compared to bone marrow. The detection was conducted in 3, 5, 7, and 9 months after the use of dasatinib, respectively. The concentrations in serum was 6.2 ± 3.41 ng/mL (3–11 ng/mL) and 3.23 ± 0.40 ng/mL (3–3.7 ng/mL) in bone marrow. Two patients with Ph+ ALL treated with imatinib for 1 month exhibited the serum imatinib concentration of 3,320 ± 183.85 ng/mL  and bone marrow Imatinib concentration of 3,020 ± 127.28 ng/mL . The concentration of imatinib in cerebrospinal fluid was 34 ng/mL in one patient. The blood-brain barrier penetration rate of imatinib was 1.07%.Firstly, the effects of imatinib, dasatinib, and flumatinib on SUP-B15 cell proliferation were detected, which all exhibited the dose—and time-dependent inhibitory effects on SUP-B15 cell proliferation. The IC50 values of SUP-B15 cells treated with three drugs were 13.25, 8.63, and 1.388 μmol/L , respectIn the control group, the nucleus of SUP-B15 was round with complete structure. The proportion of nucleus and nuclear cytoplasm was large. The cellular chromatin was abundant, indicating that the cell replication and proliferation ability were strong. In the TKI treatment group, the nuclear substance was significantly reduced, and the nuclear denaturation and shrinkage were observed. With the increase of drug concentration, the degree of nuclear shrinkage was greater, and the cytoplasm was gray blue. It is indicated that these three drugs can inhibit the proliferation of SUP B15 cell line in a dose-dependent manner .Annexin V/PI double staining was employed for apoptosis detection, which was detected as 8.89% and 7.76% after treated with 1.56 μmol/L and 0.78 μmol/L flurmatinib for 24 h, 7.55% and 6.32% with 10 μmol/L and 5 μmol/L dasatinib for 24 h, and 11.77% and 7.74% with 20 μmol/L and 10 μmol/L imatinib for 24 h. The results indicated that TKI with different concentrations could promote the apoptosis of SUP-B15 cells, with a positive dose-dependence .Due to the inhibitory effect of flumatinib on cell proliferation, the effect of flumatinib on cell cycle was detected using cell loss apparatus. The results revealed the increased proportion of G1 phase cells in SUP-B15 cells after flumatinib was treated with SUP-B15 cells, as the most obvious in 3.12 μmol/L group, suggesting the SUP-B15 block by flumatinib in G1 phase .+ ALL patients, comparable to second-generation TKI. However, the long-term survival rate of flumatinib in Ph+ ALL patients requires further observation by follow-up.A total of 29 patients with Ph+ ALL were enrolled in the present study. After the therapy of flumatinib combined with VP induction, the CR rate reached 96.3%, with 3-month CR rate of 87.5%, and 6-month of 86.7%, which showed a slight superiority to A Phase II Study of Flumatinib with Chemotherapy for Newly Diagnosed Ph/BCR-ABL1-Positive Acute Lymphoblastic Leukemia in Adults . The remOver the years, monitoring MRD has been adopted as a key element in acute leukemia management to more precisely define the depth of patient response to treatment, including Ph+ ALL . The MRDIn terms of molecular genetics, Ravandi scholars  suggesteThe remission rates of patients receiving flumatinib at first diagnosis and those changed from other TKI to flumatinib were analyzed, which revealed no statistical difference. It suggested an identify efficacy by flumatinib even on those change from other TKI due to adverse drug reactions or ABL kinase mutation. However, considering the confounding factors in the comparison of the two groups of patients, such as different combination chemotherapy, different mutant spectra, the presence of extramedullary disease or the receipt of alloHSCTs, this univariate analysis still has some limitations.The efficacy and prognosis suggested age as a significantly factor to impact the efficacy of molecular remission of the disease. In terms of prognosis, age ≥60 years old, presence of IKZF1, failure to achieve induced remission or MMR after 3 months of treatment, and the failure to undergo hematopoietic stem cell transplantation in high-risk patients will result in adverse effects on prognosis. During treatment, it mainly referred to myelosuppression, liver insufficiency and infection, and the observed reactions such as cardiotoxicity and renal impairment were generally tolerable.This study refers to a retrospective analysis depending on a single center with a relatively limited number of cases, and various confounding factors inevitably affect the results. In the later stage, it is necessary to expand the sample size and conduct a multi-center randomized controlled trial to further evaluate the difference in efficacy of imatinib, dasatinib and flumatinib in patients with Ph+ ALL.The pharmacokinetic (PK) of flumatinib indicated that flumatinib could serve as a safe and well tolerantly therapy, with the maximum blood concentration (Cmax) and AUC0-t linearly correlated to the dose range of 200–1,000 mg . StudiesIn the present study, the TKI concentrations in peripheral blood, bone marrow, and cerebrospinal fluid were collected from 11 patients receiving flumatinib, 3 patients receiving dasatinib, and 2 patients receiving imatinib and analyzed, revealing the highest by flumatinib in Ph+ ALL bone marrow, ordered as bone marrow > serum > cerebrospinal fluid. In contrast, the concentration of dasatinib and imatinib in blood was ordered as serum > bone marrow > cerebrospinal fluid. The wide distribution of flumatinib in tissues suggests a more considerable scavenging effect on intramedullary and extramedullary infiltrated leukemia cells. Meanwhile, that flumatinib could be detected in the cerebrospinal fluid of Ph+ ALL patients suggests a high probability to cross the blood-brain barrier, which requires study with the extended data considering the relatively limited number of patients in this study. The concentration of dasatinib in the cerebrospinal fluid of the patients was lower than the lowest limit of detection, which means a requirement of further study to investigate the correlation between dasatinib and the treatment of central leukemia.Pharmacodynamic analysis of Ph+ ALL cell line SUP-B15 revealed the differentiation elicited by flumatinib, as well as the promoted apoptosis of SUP-B15 cells, inhibited proliferation of Ph+ ALL cells, and the block of cells in the G1 phase. Moreover, the IC50 value reached the lowest by flumatinib in the CCK-8 compared with imatinib and dasatinib, suggesting a better effect of flumatinib.In summary, flumatinib could achieve a better remission effect on Ph+ ALL, with comparable efficacy to second-generation TKI, and resulting in tolerable adverse reactions. Cytological experiments can also support the above conclusions. In terms of drug concentration monitoring, flumatinib was revealed to penetrate the blood-brain barrier, which is worth promoting in clinic.