Background: A series of α-Mangostin (α-M) derivatives were designed and synthesized. α-M and four analogues were evaluated for their multifunctional anti-Alzheimer’s disease (anti-AD) effects on fibrillogenesis, microglial uptake, microglial degradation, and anti-neurotoxicity of Aβ, as well as LPS-induced neuroinflammation. The differences in bioactivities were analyzed to understand the structure-activity relationship for further modifications.Purpose: This study aims to investigate the anti-AD effects of α-M and elucidate its structure-activity relationship by comparing difference between α-M and several analogues.Methods: Aβ fibrillogenesis was detected by Thioflavin T fluorometric assay. The levels of Aβ1-42 and inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay. Neuron viability was examined by the CCK-8 assay. The morphology of ZO-1 of bEnd.3 cultured in BV-2-conditioned medium was evaluated by immunofluorescence staining.Results: Aβ fibrillogenesis was significantly inhibited by co-incubation with α-M, Zcbd-2 or Zcbd-3. α-M, Zcbd-2, Zcbd-3, and Zcbd-4 decreased the levels of Aβ1-42 and inflammatory cytokines, and promoted Aβ uptake, degradation and anti-inflammation effects inflammation in microglia. α-M and Zcbd-3 protected neuron viability from Aβ-induced neurotoxicity, and preserved tight junction integrity of bEnd.3 against LPS-induced neuroinflammation.Conclusion: Zcbd-3 acted as α-M almost in all effects. The structure-activity analysis indicated that the 3-methyl-2-butenyl group at C-8 is essential for the bioactivity of α-M, while modifying the double hydroxylation at the C-2 position may improve the multifunctional anti-AD effects. Alzheimer’s disease (AD), a devastating and eventually fatal neurodegenerative disease, is characterized by progressive loss of cognition and disruption of essential functions . As the 6β1 integrin  aggregation , microgl1-42 aggregation and exerts neuroprotective effects  is a polyphenolic xanthone derived from mangosteen. Recent studies demonstrated that α-M has a broader range of pharmacological properties, such as anti-cancer , antioxiis issue .in vivo application of α-M due to its hydrophobic scaffold, which leads to poor aqueous solubility and low bioavailability in the brain. Therefore, structure modification is inevitable to enhance the druggability of α-M. The structure optimization is based on knowledge about the respective influences of key modification sites on their multifunctional activities. However, only a little was known about it. To elucidate the structure-function relationship of α-M’s anti-AD activities, we investigated the effects of α-M and several representative analogues  and dissolved in hexafluoroisopropanol  at 1 mg/ml, stored at −20°C. α-M was purchased from Shanghai Sunny Biotech Co. Ltd., with a purity >99% by HPLC and dissolved in DMSO to 5 mM, stored at −20°C. The xanthone derivatives were synthesized as our previously published method (Escherichia coli 0111:B4) and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich . Dulbecco’s modified Eagle’s medium (DMEM), minimal essential medium (MEM), fetal bovine serum (FBS), Neurobasalmedium, Gluta-MAX™-I supplements, non-essential amino acids and B27 supplement were purchased from Gibco . Penicillin, streptomycin, phosphate-buffered saline , and other cell culture reagents were purchased from HyClone Laboratories . DAPI and BCA kits were purchased from Beyotime Biotechnology . Rabbit anti-Zona Occluden-1 (ZO-1) antibodies were purchased from Abcam . Alexa Fluor 647-conjugated goat anti-rabbit IgG was from Abcam . Aβ ELISA kits were purchased from Invitrogen . TNF-α and IL-6 ELISA kits were purchased from Multisciences Biotech .Aβd method . Cell co2+ and Mg2+ . Dissected pieces of cortical tissue were pooled together and transferred to an enzymatic dissociation medium containing 0.05% trypsin-EDTA  and then incubated for 6–8 min at 37°C. After enzymatic dissociation, the trypsin solution was aspirated, and the tissue was triturated with a sterile Pasteur pipette in complete Minimum Essential Medium , 5% horse serum (v/v), 2 mM L-glutamine, 100 units/mL penicillin, 100 mg/ml streptomycin) plus 2000 IU/ml DNase. After 800 g for 8 min centrifugation, the pellets were resuspended in complete MEM medium and plated on poly-L-lysine pre-coated 96-well or 24-well plates. Cells were cultured in a humidified atmosphere (5% CO2—95% room air) at 37°C for 2 h, then the medium was replaced with neurobasal medium plus 2% B27 and 2 mM Gluta-MAX™-I supplements. The purity of neurons is around 90%, as indicated by Hoechst staining for nucleus and β-tubulin staining for neural cell body and neurites.With mild modifications, primary mouse cerebral cortical neurons were prepared as previously described . Neurons3, 4 mM glutamine, 10% FBS, 100 μg/ml penicillin, and 100 μg/ml streptomycin. Cells were grown in a humidified atmosphere containing 5% CO2 at 37°C and passaged every 2–3 days. When reaching the required confluence, cells were changed to serum-free medium prior to drug treatment.The murine microglial cells BV-2 (3111C0001CCC000063) was obtained from the National Platform of Experimental Cell Resources for Sci-Tech (4), and cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/ml streptomycin, 1% Gluta-MAX™-I supplements and 1% non-essential amino acids. The murine mouse brain endothelial cell line bEnd.3 (ATCC CRL2299) was obtained from ATCC and cultured in DMEM with 0.45% glucose, 0.37% NaHCO2O, the α-M stock was diluted in 0.1 M TBS with 0.02% Tween 20, and ThT was dissolved in 50 mM Glycine-NaOH (pH 8.5).The fibril formation of the Aβ peptides was measured by Thioflavin T (ThT)  fluorometric assay according to published reports with some modifications . Aβ was 2, Zcbd-3, Zcbd-4, and Zcbd-5, Aβ1-40 (1 μg/ml) was mixed with α-M or its analogues in 1:1 molar ratio and incubated for 30 min. After incubation, ThT was added to each test sample to a final concentration of 10 μM and set at 37°C. The ThT fluorescence was monitored at an excitation of 446 nm and an emission of 485 nm with a bandwidth of 5 nm, and the measurement was continued for 24 h at 30 min intervals using Varioskan Flash plate reader .To evaluate 24 h-inhibition of Aβ fibril formation by α-M, Zcbd-1-40 (100 mg/ml) was incubated for 2 days at 37°C to generate fibrils. Then the preformed fibrils were mixed with an equal molar amount of α-M or its analogues for 8 h at 37°C. After incubation, ThT was added to each test sample to a final concentration of 10 μM. The ThT fluorescence was monitored at an excitation of 446 nm and an emission of 485 nm with a bandwidth of 2.5 nm, and the measurement was continued for 10 min at 1 min intervals using Varioskan Flash plate reader .To monitor the disaggregation of preformed fibrils, AβBV-2 cells were pre-treated with α-M (250 nM) or its analogues (100 nM) for 24 h. Then the BV-2 cells were incubated for an additional 4 h in DMEM with a mixture of 1 μg/ml Aβ and α-M (250 nM) or its analogues (100 nM). Afterwards, cells were extensively washed with PBS and were lysed in cell lysis buffer  supplemented with EDTA-free protease inhibitors (Roche). Lysates were centrifuged at 12,000 g at 4°C for 15 min. Supernatants were collected and further applied for ELISA. Aβ levels were measured using Aβ ELISA kit (Wako) and normalized to total protein concentration assessed by the BCA method (Pierce).BV-2 cells were pre-treated with α-M (250 nM) or its analogues (100 nM) for 2 h and then stimulated by LPS (100 ng/ml) for 24 h. Then the medium from each group was collected. According to the manufacturer’s instructions, the concentrations of TNF-α and IL-6 in the cell culture media were determined using commercially available ELISA kits . The concentration was calculated from a standard curve obtained by adding sodium nitrite to DMEM.1–42 oligomer, the dried monomeric peptide Aβ was resolved in ddH2O at a concentration of 100 mM and incubated at 4°C for 24 h. The cell viability of primary cultured cerebral cortical neurons with Aβ oligomers or α-M/Zcbd-3 was measured by the CCK-8 kit . Cells were seeded onto 96-well plates at a density of 1 × 104 cells per well and allowed to grow to the required confluence. On day 7 after culturing, Aβ1-42 oligomers and α-M/Zcbd-3 were added directly to reach the final concentrations of 1 μM for Aβ1-42 oligomers and 0.5, 5, and 50 nM for α-M/Zcbd-3. Then the cells were incubated for 24 h, and cell viability was measured with a CCK-8 kit according to the manufacturer’s instructions. Cells were incubated at 37°C for 1 h; the absorbance was read by Varioskan Flash plate reader  at 450 nm.For the preparation of Aβ5 per dish. After treatment, they were fixed with 4% paraformaldehyde for 1 h, permeabilized by 0.4% Triton X-100 in PBS, and blocked in PBST supplemented with 5% sheep serum for 30 min at room temperature. Afterwards, they were incubated with rabbit anti-ZO-1 (1: 400) antibody overnight at 4°C. After PBS washing, they were incubated with an Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (1:1000) for 1 h at room temperature. Then cells were counterstained with DAPI for 10 min. Images were captured with a Leica TCS SP8 confocal microscope  and further analyzed using Image J and Adobe Photoshop CC 2019.Mouse brain endothelial bEnd.3 cells were seeded onto 35-mm dishes at a density of 2 × 10p < 0.05 taken to indicate significant difference.Data were presented as mean ± standard error of the mean (SEM). They were analyzed by student’s T-test or one way-ANOVA followed by Dunnett’s test using GraphPad Prism 8 statistical analysis software, with 1-40 co-incubated with α-M or its analogues all kept at a low level, which indicated that α-M and its analogues significantly inhibited the formation of Aβ1-40 fibrils . As shown in 2 at a concentration of 100 nM, 1 μM, and 10 μM. In 3, confirming that both Zcbd-2 and Zcbd-3 could induce the disaggregation of pre-aggregated Aβ1-40. In addition, the results in 2, indicating that Zcbd-2 possessed a dose-dependent promotion on Aβ fibrils disaggregation. In contrast, low concentrations of Zcbd-3 kept little inhibitory effect on Aβ aggregation, yet it displayed a 2.75-fold higher disaggregation efficacy than Zcbd-2 at a high concentration of 10 μM  decreased cell viability by 29.83% of cortical neurons, while co-incubation of Aβ1-42 with 5 nM and 50 nM α-M significantly reversed the viability to 80.27% ± 1.69% and 83.02 ± 5.54%, respectively. According to the analysis above, the EC50 of α-M’s neuroprotection from Aβ1-42 oligomers was 0.70 nM. Meanwhile, Zcbd-3 of equal concentration also rescued the amount of living neuronal to 84.50 ± 3.13% and 84.61 ± 5.79%.Aβ oligomers have been confirmed to be neurotoxic through inducing dendritic simplification, synaptic dysfunction, dystrophic neurites and cell apoptosis. To precisely evaluate the protective effects of α-M and Zcbd-3 before the administration of LPS remarkably rescued the expression of ZO-1 in bEnd.3 by 66.58% (α-M) and 77.31% (Zcbd-3), compared to the group without pretreatment. Meanwhile, as shown in 3. According to the increase in expression and distribution of ZO-1, we found that α-M and its analogue contribute to maintaining the morphological integrity of BBB affected by neuroinflammation. The data revealed that Zcbd-3, as well as α-M, possessed a strong inhibitory effect on the LPS-induced microglial neurotoxicity and exerted protection of brain structures.Disruption of tight junctions (TJs) is a hallmark of AD, which enhances the permeability of BBB. Overactivated BV-2 by neuroinflammation may release cytokines to destroy the nervous system in a few ways, including BBB dysfunction. bEnd.3 mouse brain endothelial cell line keeps the characteristic of BBB as a functional barrier. Thus it is an ideal model for BBB research. In order to monitor an AD-anastomotic brain environment, BV-2 cells were stimulated by LPS  to simulate inflammation response in AD patients’ brains. Then the BV-2-conditioned media was transferred to bEnd.3 cells to observe the effect of microglia-mediated neuroinflammation on BBB. As an intracellular scaffold protein, the numerical and spatial changes of ZO-1 reflect the integrity and functional status of BBB. Thus we detected the expression and distribution of ZO-1 in bEnd.3 mouse brain endothelial cells after 24 h-treatment with BV-2-conditioned media. The immunofluorescence staining results showed that the pretreatment with α-M or Zcbd-The most widely accepted hypothesis for the pathogenesis of AD considers Aβ to play a crucial role in AD’s development and progression . A steadIn AD, Aβ aggregates to form plaques and tangles, which comprise the histopathological hallmarks . Early eAlthough the present clinical treatment could decrease the level of Aβ in blood and cerebrospinal fluid and clear Aβ deposits in the brain , AD patiNatural products with diverse structures were pivotal sources for novel lead compounds in drug discovery. Our previous studies proved that α-M, one of the natural products, displayed protective effects on Aβ aggregation and inflammation-induced neurotoxicity. Because of its multifunctional anti-AD effects, α-M attracted our interest in anti-AD drug discovery. Nevertheless, several restrictions may hamper its extensive use, such as low solubility, absorption limitation, and metabolic stability. Structural modification is an inevitable way for α-M to achieve a final druggable candidate. Therefore, we designed a series of α-M analogues to understand better its structure-activity relationship, especially which part is applicable for modification.In this study, we demonstrated the effects of α-M and its four analogues on inhibiting Aβ fibrillogenesis, disaggregating preformed Aβ fibrils, accelerating Aβ clearance, protecting neurons from LPS-induced neuroinflammatory response, anti-neurotoxicity, and preserving the integrity of tight junctions.2 and Zcbd-3, significantly disaggregated preformed Aβ. Pharmacodynamic correlation between dosage and activity is one of the prerequisites for satisfactory druggability. Thus we designed the co-incubation of Aβ and Zcbd-2 and Zcbd-3 of three different concentrations  to investigate the disaggregation of Aβ by co-incubation with Zcbd-2 and Zcbd-3. The fluorescence signals reflected that both Zcbd-2 and Zcbd-3 significantly affected the disaggregation of preformed Aβ fibrils in a concentration-dependant manner. The ThT results also suggested that all four derivatives have no negative influence on the Aβ fibrillogenesis inhibitory activity of α-M. Moreover, the modifications in Zcbd-2 and Zcbd-3 retained the disaggregating effect of α-M on preformed fibrils.The ThT fluorescence assay results revealed that α-M and the analogues inhibited the formation of Aβ fibrils during 24 h co-incubation. In addition, two of α-M’s analogues, Zcbd-3 significantly elevated the efficiency of Aβ uptake and degradation in microglia, while Zcbd-2 and Zcbd-4 displayed little effect on the promotion of Aβ clearance. The results indicated that the variable sites in Zcbd-3 were applicable for further structural modifications, meanwhile the structural changes in Zcbd-2 and Zcbd-4 may affect the ability of microglia to uptake and degrade Aβ.There are several widely-recognized Aβ clearance pathways in the central nervous system, including interstitial drainage, cerebrospinal fluid, and proteolytic digestion. As innate immune cells in central nervous system, microglia help maintain neural environment homeostasis, support regeneration and induce repair through clearing debris and aberrant proteins, including Aβ plaques . Any fun1-42 has been found to possess a greater propensity to aggregate compared to Aβ1-40, and it is the predominant component of AD plaques (1-42 oligomers-induced neurotoxicity is well-considered to be beneficial during such a slowly progressive and highly dynamic process in AD. To evaluate the protective effects of α-M and its analogues on Aβ-induced neurotoxicity, we treated α-M or its analogues-pretreated primary cerebral cortical neurons with Aβ1-42 oligomers. Cell viability after 24-h incubation was manifested by the CCK-8 assay, which demonstrated that co-administration of α-M or Zcbd-3 significantly elevated the cell viability depressed by neurotoxic Aβ oligomers. The analysis results indicated that α-M and Zcbd-3 remarkably attenuated the neurotoxicity of Aβ oligomers and exerted neuroprotective effects in cortical neuron cells.Due to its larger surface area directly interacting with the neuronal membranes, it is now proposed that, compared to Aβ fibrils, Aβ oligomers are easier to diffuse into synaptic clefts, cause the impairment of neuronal signal transmittion, and result in the neurological decline characteristic of AD . Moreove plaques . It inte plaques . In the  plaques , directl plaques . Thus al4 displayed more potent effects on inhibiting the release of the pro-inflammatory cytokines than α-M, which suggested that the change in Zcbd-4 itself is a modification to enhance the anti-inflammatory effect of α-M.In addition to Aβ deposition, microglia-mediated neuroinflammation also induces neuronal injury and memory dysfunction in AD . Inflamm3 on Aβ oligomers-induced neurotoxicity, we defected their effects on BBB dysfunction due to LPS-induced inflammatory response by evaluating the expression and morphology of ZO-1 in bEnd.3 cerebral microvascular endothelial cells. The immunofluorescence staining of ZO-1 illustrated that bEnd.3 pre-treated by α-M or Zcbd-3 appeared to be closer to the normal status, which indicated the protective effects of both α-M and Zcbd-3 on keeping the homeostasis of NVU.Microglia also works as the first line guardian against BBB injury in the central inflammation response . BBB is 3 shared the hightest anti-AD activity in general; while Zcbd-2 and Zcbd-4 displayed weaker ability in lessening Aβ deposition, and Zcbd-5 had a lower comprehensive effect. Specifically, α-M possessed a better performance on inhibiting Aβ fibrillogenesis, disaggregation, uptake and degradation than Zcbd-2, which was structurally modified at the C-8 position. This result indicated that the structure modification at the C-8 position might harm the activity of α-M. Compared to Zcbd-2, Zcbd-3 exerted more substantial effects on Aβ disaggregation, microglial uptake and microglial degradation, and displayed similar features as α-M in all assessments.For α-M contains two 3-methyl-2-butenyl groups at C-2 and C-8 positions as potential functional groups, we synthesized four analogues to evaluate whether they are modifiable sites. These derivates share the same structural scaffold with α-M; meanwhile, the two 3-methyl-2-butenyl groups were modified differently. As shown in 2 and Zcbd-3 was the chemical groups at the C-2 position, and we hypothesized that it was a potential modification site. Moreover, the introduction of hydroxyl at the C-2 position counteracted the activity loss of the structural modification at the C-8 position, which suggested that the double hydroxylation at the C-2 position may enhance the multifunctional bioactivities of α-M. Besides, compared to Zcbd-3, the active effects of Zcbd-4 and Zcbd-5 were attenuated, which indicated that different modifications at the C-2 position would have different consequences for the bioactivity of α-M. Also, although Zcbd-2, Zcbd-4 and Zcbd-5’s all-around performance was not as satisfactory as Zcbd-3, their performance in a specific aspect could be even better than α-M.On account of that, the main difference between Zcbd-In conclusion, our study demonstrated the multifunctional features of α-M and its analogues, beneficial to Aβ fibrillogenesis, disaggregation, uptake and degradation, Aβ oligomer induced neurotoxicity, LPS induced neuroinflammation, and the integrity of tight junctions. It was found that the 3-methyl-2-butenyl group at C-8 is essential for the bioactivity of α-M, while modifying the double hydroxylation at the C-2 position may improve the multifunctional anti-AD effects. These structural insights may help us better understand the structure-activity relationship and accelerate α-M modification in drug development.


II ion has an N2O2 square-planar coordination sphere defined by two N atoms and two O atoms of the tetradentate dianionic 4,4′-dibromo-2,2′-diphenolato ligand.The central Ni 20H18Br2N2O2)], the NiII ion is four-coordinated in a slightly distorted square-planar coordination geometry defined by two N atoms and two O atoms of the tetradentate dianionic 4,4′-dibromo-2,2′-diphenolato ligand. Pairs of complex molecules are assembled by intermolecular C—H⋯O hydrogen bonds with d(C⋯O) = 3.247 (4) Å.In the title compound, [Ni(C The tignd Fig. 1, pairs ond Fig. 1. In addi2  in acetone (30 ml) was added 4,4′-dibromo-2,2′-diphenol −3) and the deepest hole (−0.51 e Å−3) in the difference Fourier map are located 0.86 and 0.70 Å, respectively, from the atoms Br1 and Br2.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314621000845/sj4220sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314621000845/sj4220Isup2.hklStructure factors: contains datablock(s) I. DOI: 2058387CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Crystal growth of 3′-carboxy-3-methyl--1-ium chloride in ethanol led to single crystals of ethyl 4-oxo-1,4-dihydropyridine-3-carboxylic acid, which likely resulted from hydrolysis and esterification of the original compound, and its crystal structure was determined by single-crystal X-ray diffraction. 8H9NO3, likely generated through hydrolysis and esterification of 3′-carboxy-3-methyl--1-ium chloride by ethanol, which contained water, has a nearly planar conformation. The crystal structure is sustained by one-dimensional chains along the a-axis direction based on bifurcated N—H⋯ hydrogen bonds between the NH group of the 4-oxo-1,4-dihydropyridine ring and the two carbonyl O atoms.The title compound, C The hydrogen bond parameters for NH⋯O=C (ring) are: 1.96 (2) Å for bond length, and 134.9 (17)° for the bond angle. The corresponding parameters for NH⋯O=C (ester) are 2.15 (2) Å and 139.6 (17)° ° Fig. 2.The title compound was obtained during an attempt to grow single crystals of 3′-carboxy-3-methyl--1-ium chloride by slow evaporation of an ethanolic solution. 3′-Carboxy-3-methyl--1-ium chloride was dissolved in bulk ethanol at 343 K, and then the resulting solution was left in a refrigerator. Colorless plate-shaped crystals Fig. 3 were harCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314621005551/pk4034sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314621005551/pk4034Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621005551/pk4034Isup3.cmlSupporting information file. DOI: 2086773CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Different from Fe ions in Fenton reaction, Mn ions can function both as catalyst for chemodynamic therapy and immune adjuvant for antitumor immune responses. In Mn-mediated Fenton-like reaction, bicarbonate (3-ICG) were designed for intracellular marriage of bicarbonate and Mn ions as “immune ion reactors” to regulate intracellular redox homeostasis and antitumor immune responses. Under the tumor acidic environment, the biodegradable complex can release “ion reactors” of Mn2+ and 2+-enhanced immunotherapy effectively suppress tumor growth and realize complete tumor elimination.Herein, Stimuli-responsive manganese carbonate-indocyanine green complexes (MnCOThe combination therapy strategy with the help of novel immune adjuvants would produce an enhanced immune response, and be used for the treatment of deep tumors in situ.The online version contains supplementary material available at 10.1186/s12951-022-01404-x. After centrifugation, the MB (10 μg/mL) and H2O2 (1 mM) were added to the supernatant. The absorbance of MB was measured after shaking at 37 °C for 30 min.MnCO3-ICG ([Mn]: 50 μg/mL) were incubated with different different concentrations of NaHCO3 in the acidic environment for 30 min. After centrifugation, the MB (10 μg/mL) and H2O2 (1 mM) were added to the supernatant. The absorbance of MB was measured after shaking at 37 °C for 30 min.MnCO3-ICG and Mn-ICG ([Mn]: 50 μg/mL) were incubated with different buffer solution  for 30 min. After centrifugation, the MB (10 μg/mL) and H2O2 (1 mM) were added to the supernatant. The absorbance of MB was measured after shaking at 37 °C for 30 min.MnCO3-ICG ([Mn]: 50 μg/mL) were incubated in the acidic environment (pH 5.8) for different time. After centrifugation, the MB (10 μg/mL) and H2O2 (1 mM) were added to the supernatant. The absorbance of MB was measured after shaking at 37 °C for 30 min.MnCO5 cells for 24 h. After that, the cells incubated with MnCO3-ICG ([Mn]: 5 μg/mL) in the dark for different time . After washing with PBS for three times, the cells were stained with hoechst33342 (5 μg/mL) and imaged by a laser scanning confocal fluorescence microscope.To evaluate the cell uptake efficiency, 4T1 cells were seeded in observation dish at a density 105 cells for 24 h. After that, the cells incubated with MnCO3-ICG ([Mn]: 5 μg/mL) in the dark for different time . After washing with PBS for three times, the cells were digested and analyzed by flow cytometer (ACEA Biosciences).For quantitative analysis the cell uptake efficiency, 4T1 cells were seeded in six-well plates at a density 105 cells for 24 h, and incubated with MnCO3-ICG ([Mn]: 5 μg/mL) in the dark for 24 h. After washing with PBS for three times, the cells were stained with hoechst33342 (5 μg/mL) and lyso-tracker (2 μM). The stained cells were imaged by a laser scanning confocal fluorescence microscope.4T1 cells were also seeded in observation dish at a density 104 per well) and incubated with different concentrations of MnCO3-ICG for 24 h or 48 h. Cells viability were determined by the MTT assay. The photothermal therapy effect was detected by exposing cells to NIR laser  for 5 min. Cells viability were also determined by the MTT assay.To evaluate the chemodynamic therapy effect, 4T1 cells, U87MG cells and Hep G2 cells were seeded in 96-well plates  for 24 h. The photothermal therapy group were exposed to NIR laser  for 5 min. After that, the cells were continue incubated for another 24 h, and staining with AM/PI dual-staining kit. After washing with PBS for three times, the cells were imaged by an inverted fluorescence microscope.To visually observe the living and dead cells, 4T1 cells were seeded in six-well plates at a density 103-ICG ([Mn]: 20 or 50 μg/mL) were added to the dishes of the MCSs for 24 h. Then, the photothermal therapy group were exposed to NIR laser  for 5 min. The MCSs were continue incubated for another 24 h, and staining with AM/PI dual-staining kit. After washing with PBS for three times, the MCSs were imaged by a laser scanning confocal fluorescence microscope.4T1 cells (3000 cells per well) were seeded into 96-wells plate containing 1% agarose to form MCSs. The MnCO3 in average volume, the mice were randomly divide d into 6 groups: (Group 1) PBS (four doses); (Group 2) MnCO3-ICG ; (Group 3) 2 × MnCO3-ICG ; (Group 4) 4 × MnCO3-ICG ; (Group 5) MnCO3-ICG ([Mn]:2 mg/kg + L (0.5 W/cm2), one dose, termed as PTT); and (Group 6) MnCO3-ICG ([Mn]:2 mg/kg + L (0.5 W/cm2), three doses, termed as PTT + CDT). The laser  was applied to tumor at 4 h post-injection of MnCO3-ICG. The body weight and tumor size of the mice were recorded for the next 14 days. The relative tumor volume of each mouse was acquired by dividing by the initial tumor volume Tumor volumes and body weight were also recorded. After 14 days, the mice were euthanized and the collected tumor and major organ were kept for H&E staining.When the tumor reached 60 mmIL-6 (Elabscience), TNF-α (Elabscience) and IL-1α (Elabscience) in mouse serum samples after different treatment were analyzed with ELISA kits according to the vendors’ protocols.To analyze immune cells by flow cytometry, spleens of mice after various treatments were collected and stained according to the manufacturer’s protocols. In brief, to analyze memory T cells, cells from tumors spleens were stained with antibodies against CD8a-APC  and CD3-PE . To analyze the maturation of DCs, cells from tumors spleens were stained with antibodies against CD80-APC , CD86-PE , CD11c-FITC .The contrast between each group were analyzed by the Student’s t test and the nonparametric test to assess statistical significance . All experiments were performed at least three times and expressed as means ± SD.3-ICG complexes were prepared through a gas diffusion procedure, which are monodisperse with an average diameter of 72 nm  , and cell culture medium (DMEM)  to Mn(IV), after being dissociated and re-assembled. The decomposition profiles of MnCO3-ICG were measured using MR and US phantom investigations. Under mildly acidic condition, the Mn2+ ion release increased gradually and then increased sharply with existing H2O2 and GSH, indicating the time-dependent T1 intensity change ; a rapid degradation of MB was found by H2O2 plus 3-ICG by electron paramagnetic resonance (EPR) spectroscopy. In comparison with H2O2 alone, obvious characteristic peaks (1:2:2:1) were recognized in Mn2+/3-ICG played a beneficial role. Furthermore, the absorbance of MB dropped sharply by increasing :2 mg/kg + L (0.5 W/cm2), one dose, termed as PTT); and (Group 6) MnCO3-ICG ([Mn]:2 mg/kg + L (0.5 W/cm2), three doses, termed as PTT + CDT). The laser  was applied to the tumors at 4 h post-injection of MnCO3-ICG. We found that the tumors on the mice of Groups 4, 5 and 6 were more effectively suppressed over those in the other Groups  versus those with no treatment  activating innate immunity :2 mg/kg + L (0.5 W/cm2), three doses, termed as PTT + CDT). Three injections were administered intravenously on days 0, 3 and 6, and the laser  was applied to the tumors at 8 h post-1st-injection of MnCO3-ICG. During the treatment, the orthotopic hepatic tumor growth was monitored by BLI. The fluorescence intensity of group 3 remained unchanged or even decreased slightly, which was a sharp contrast to the increase of fluorescence intensity in the control group -enhanced immunotherapy effectively suppressed the tumor growth, and realized complete tumor elimination. The combination therapy strategy promotes DC maturation, enhances the CTL-mediated cytotoxic effect, and generates multiple pro-inflammatory factors . This study provides a potential synergistical strategy to improve the therapeutic effect for orthotopic tumors by releasing “ion drug” in an on-demand manner.In summary, we proposed a self-supplying bicarbonate- and Mn-pool Fenton-like MnCOAdditional file 1: Figure S1. Typical TEM images of MnCO3-ICG complexes prepared at different feeding ratios of ICG and MnCl2·4H2O. Figure S2. (a) the absorption spectra of MnCO3-ICG complexes at different time. (b) Typical TEM images of MnCO3-ICG complexes prepared at different time. Figure S3. (a) TEM imaging of MnCO3-ICG-PAH complexes prepared with different feeding ratios of MnCO3-ICG and PAH. (b) Photographs of MnCO3-ICG-PAH complexes after being incubated in H2O, PBS (7.4), cell medium (DMEM), and serum (FBS) for 12 h. (c) The hydrodynamic diameter of MnCO3-ICG-PAH complexes with different feeding ratios. Figure S4. (a) X-ray powder diffraction pattern of the MnCO3-ICG and the re-assembled nanoformulation after incubation in the acidic buffer solutions. (b) XPS spectra for MnCO3-ICG and the re-assembled nanoformulation after incubation in the acidic buffer solutions. XPS spectra of Mn2p for (c) for MnCO3-ICG and (d) the re-assembled nanoformulation after incubation in the acidic buffer solutions. Figure S5. (a) Ultrasound images of the generation of CO2 after CaCO3 incubation with different time (pH 5.8 + H2O2). (b) Ultrasound signal intensity of the generation of CO2 after CaCO3 incubation with different time (pH 5.8 + H2O2). (c) The typical chromatograms of CO2 generated after MnCO3-ICG incubation with acidic environment. Figure S6. (a) Colorimetric analysis of the Fenton-like reaction for MB decolorization with different concentration of HCO3− in acidic environment. (b) Colorimetric analysis of the Fenton-like reaction for MB decolorization with different treatment. (c) Colorimetric analysis of the Fenton-like reaction for MB decolorization after different reaction time with acidic environment (pH 5.8). Figure S7. (a) Colorimetric analysis of the Fenton-like reaction for MB decolorization after pulling a vacuum. (b) Bar plot showing the of MB after different treatments. Figure S8. The absorption spectra of ICG, Mn-ICG and MnCO3-ICG. Figure S9. (a) Hep G2 cells and (b) U87MG cells incubated with different concentrations of MnCO3-ICG with or without 808 nm laser  irradiation. Figure S10. Relative cellular viabilities of normal L02 and 3T3 cells incubated with different concentrations of MnCO3-ICG. Figure S11. Intracellular ·OH generation after incubation with Mn-ICG detected by DCFH-DA probe . Figure S12. Lipid damage assessment measured by lipid peroxidation assays (*p < 0.05). Figure S13. (a) CLSM observation on the intracellular distribution of lipoperoxides in 4T1 cells after incubation with PBS and Mn-ICG for 24 h. The red fluorescence is the lipid ROS ROS in cells and membranes after the staining with BODIPY-C11 . (b) lipoperoxides, based on BODIPY staining resuts in panel (a). Figure S14. (a) CLSM observation on the changes in the mitochondrial membrane potential of 4T1 cells after incubation with different concentration of Mn-ICG . (b) The membrane potential (ΔΨm) changes, assessed by JC-1staining. Figure S15. LDH release assay after incubation with different concentration of Mn-ICG. Figure S16. T1-relaxation rate (r1) and T1-weighted MR images of MnCO3-ICG. Figure S17. Ex vivo fluorescence images of the organs harvested in BALB/C tumor-bearing mice at 24 h post-injection. Figure S18. Body photos in different formulations after the 14-day treatment period. Groups 1, 2, 3 and 4 were used to represent PBS, MnCO3-ICG , 2 × MnCO3-ICG , 4 × MnCO3-ICG , respectively. Figure S19. Images of representative tumors taken from mice in different formulations after the 14-day treatment period. Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG , 2 × MnCO3-ICG , 4 × MnCO3-ICG , MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respective. Figure S20. The tumor growth inhibition curves of BALB/C tumor-bearing mice exposed to different formulations after the treatment period. Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG , 2 × MnCO3-ICG , 4 × MnCO3-ICG , MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respective. Figure S21. The T2-MR imaging and digital photos of mice BALB/C tumor-bearing after the 21-day treatment period (group 6). Groups 6 was used to represent MnCO3-ICG([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respectively. Figure S22. Representative immunofluorescence staining of nucleus (blue) and vessel (green) on the tumor slices collected 14-day after laser irradiation . Figure S23. Representative immunofluorescence staining of TUNEL (nucleus (blue) and apoptotic cells (red)) and ROS (nucleus (blue) and ROS (green)) on the tumor slices. . Figure S24. The flow cytometric histograms showing the intratumor infiltration of the effector T cells (CD8 + T cells). Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG , 2 × MnCO3-ICG , 4 × MnCO3-ICG , MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respectively. Figure S25. Body weight curves of BALB/C tumor-bearing mice after various treatments (n = 5). Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG , 2 × MnCO3-ICG , 4 × MnCO3-ICG , MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respectively. Figure S26. Hematoxylin & eosin (H&E)-stained of the organ harvested from mice in different formulations after the 14-day treatment period. Groups 1, 2, 3, 4, 5 and 6 were used to represent PBS, MnCO3-ICG , 2 × MnCO3-ICG , 4 × MnCO3-ICG , MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), one dose) and MnCO3-ICG ([Mn]: 2 mg/kg + L (0.5 W/cm2), three dose), respectively, . Figure S27. (a) Intracellular ·OH generation after incubation with MnCO3-ICG in different buffer solutions  detected by DCFH-DA probe. The blue and green fluorescence indicate cell nucleus and DCFH-DA, respectively . (b) The fluorescence intensity of ·OH,based on DCFH-DA staining resuts in panel (a) (**P < 0.05). Figure S28. (a) CLSM observation on the intracellular distribution of lipoperoxides in Hep 1–6 cells after incubation with PBS and MnCO3-ICG for 24 h. The red fluorescence is the lipid ROS in cells and membranes after the staining with BODIPY-C11 . (b) lipoperoxides, based on BODIPY staining results in panel (a) (***P < 0.001). Figure S29. (A) CLSM observation on the changes in the mitochondrial membrane potential of Hep 1–6 cells after incubation with different concentration of MnCO3-ICG . (B) The membrane potential (ΔΨm) changes, assessed by JC-1staining (***P < 0.001). Figure S30. LDH release assay of Hep 1–6 cells after incubation with different concentration of MnCO3-ICG (***p < 0.001).


Saccharomyces cerevisiae, the pre-mRNA leakage 39-kDa protein (ScPml39) was reported to retain unspliced pre-mRNA prior to export through nuclear pore complexes (NPCs). Pml39 homologs outside the Saccharomycetaceae family are currently unknown, and mechanistic insight into Pml39 function is lacking. Here we determined the crystal structure of ScPml39 at 2.5 Å resolution to facilitate the discovery of orthologs beyond Saccharomycetaceae, e.g. in Schizosaccharomyces pombe or human. The crystal structure revealed integrated zf-C3HC and Rsm1 modules, which are tightly associated through a hydrophobic interface to form a single domain. Both zf-C3HC and Rsm1 modules belong to the Zn-containing BIR (Baculovirus IAP repeat)-like super family, with key residues of the canonical BIR domain being conserved. Features unique to the Pml39 modules refer to the spacing between the Zn-coordinating residues, giving rise to a substantially tilted helix αC in the zf-C3HC and Rsm1 modules, and an extra helix αAB′ in the Rsm1 module. Conservation of key residues responsible for its distinct features identifies S. pombe Rsm1 and Homo sapiens NIPA/ZC3HC1 as structural orthologs of ScPml39. Based on the recent functional characterization of NIPA/ZC3HC1 as a scaffold protein that stabilizes the nuclear basket of the NPC, our data suggest an analogous function of ScPml39 in S. cerevisiae.In  Saccharomyces cerevisiae) and mediate the transport of a tremendous range of cargoes such as water, ions, small molecules, proteins, and ribonucleoparticles across the nuclear envelope4. As exclusive transport channels between the nucleus and cytoplasm, NPCs are ideally positioned to serve as gateways or checkpoints in the flow of information from DNA to protein and have therefore long been postulated to function in “gene gating”5. NPCs indeed play key roles beyond nucleocytoplasmic transport, such as in genome organization and integrity, gene regulation, and mRNA quality control (QC)12.Nuclear pore complexes (NPCs) are large macromolecular assemblies -binding protein ScNab2, which directly binds to the C-terminal region of Myosin-like protein 1 (ScMlp1) of the nuclear basket, monitors proper 3′-mRNA processing22. In turn, ScMlp1 deletion triggers cytoplasmic leakage of intron-containing pre-mRNAs23. A similar role in the nuclear retention of unspliced reporter mRNAs in vivo was assigned to homologs in fission yeast (SpNup211) and mammals (Tpr)26. Furthermore, a pre-mRNA leakage phenotype was observed for the deletion of an ScMlp1/ScMlp2-interacting protein termed S. cerevisiae pre-mRNA leakage protein 39-kDa (ScPml39)27. Conversely, its overexpression traps intron-containing mRNAs in nuclear foci enriched in ScMlp1 and ScNab227. ScPml39 is required for cell growth in the absence of a functional Y-complex, an essential NPC building block required for mRNA export29, and is recruited to the nuclear basket of NPCs by virtue of interactions with the N-terminal regions of ScMlp1 and ScMlp227. While ScMlp1/2 and ScNab2 sequences and functions are highly conserved across phyla30, the ScPml39 sequence is unique to Saccharomycetaceae  are discarded to avoid detrimental effects on protein homeostasis or RNA metabolismeae Fig. . To dateScPml39 and identifying key residues involved in its structural integrity is a course of action to find ScPml39 orthologs31. The structure prediction programs Phyre2 and SWISS-MODEL33 identified two Baculoviral Inhibitor of apoptosis (IAP) Repeat (BIR) domains in ScPml39. BIR domains contribute to protein–protein interactions in both apoptotic38 and non-apoptotic pathways41. The canonical BIR domain is well-studied, with currently 312 structures available in the Protein Data Bank (PDB), including structures in complex with substrate peptides. Their structures are highly conserved, as illustrated by a root-mean square deviation (RMSD) of Cα atoms of less than 1.0 Å for the representative BIR domains human neuronal apoptosis inhibitor protein (PDB: 2VM5), human Survivin (PDB: 3UED), and D. melanogaster IAP1-BIR1  and one β-sheet formed by three anti-parallel strands . The BIR domain harbors a cysteine-cysteine-histidine-cysteine (CCHC)-type zinc finger (ZnF) motif with the consensus sequence Rxx(S/T)Ω…GΩ…C-x2-C-x16-H-x6-C-x-Ω (x denotes any amino acid residue and Ω an aromatic residue) Ω is located in helix αA, and the Arg residue is essential. The motif GΩ is located between αB and β1 to form a sharp β-turn. The first and second Zn-coordinating cysteine are in the loop between β2 and β3, the Zn-coordinating histidine is in helix αC, and the last Zn-coordinating cysteine is in the loop between helices αC and αD. The final aromatic residue Ω is located in helix αD to undergo π–cation interactions with Arg in the Rxx(S/T)Ω motif of helix αA  Fig. a. The im αA Fig. a.ScPml39, the two potential ZnF motifs deviate from the consensus sequence by a shortened linker between the Zn-coordinating histidine and cysteine residues: C-x2-C-xn-H-x3-C. This difference is a hallmark of the zf-C3HC (ID: PF07967) and Rsm1 (ID: PF08600) protein families, which together with the canonical BIR domain (ID: PF00653) comprise the CCHC ZnF motif-containing clan of BIR-like domains (ID: CL0417) according to the Pfam database44. ScPml39 is predicted to have two zfC3HC and/or Rsm1 domains. In contrast to the canonical BIR and zf-C3HC domains, the Rxx(S/T)Ω motif could not be identified in the Rsm1 family by a Hidden Markov model (HMM)45 45 Fig. . FurtherScPml39 and identify orthologs in S. pombe and human based on structure-guided sequence alignment. The distinct spacing between the CCHC ZnF-coordinating residues results in features that are different from the canonical BIR domain. Two zf-C3HC and Rsm1 modules tightly associate to form a novel “Pml39 fold”.Here we present the 2.5 Å-resolution crystal structure of ScPml39 was determined by limited proteolysis using the full-length recombinant protein ScPml39 (residues 1–334). Elastase digest yielded a stable single fragment of ~ 30 kDa molecular weight   with a dissociation constant (KD) of ~ 13 µM and a 1:1 molar ratio, as measured by isothermal titration calorimetry  (Table ScPml3977–317 contains two BIR-like modules: zf-C3HC (residues 79–189 in blue) and Rsm1 (residues 190–311 in purple) , consistent with the limited proteolysis data  Table . The cryScPml39 zf-C3HC module comprises five α-helices (αA (83–98), αB (111–117), αC (156–171), αD (177–180), and αE (185–189)) and one antiparallel β-sheet composed of three strands (β1 (119–121), β2 (129–134), and β3 (138–144)) ) Fig. . Arg89 a) Fig. . ule Fig. . Cys134,tif Fig. , S2. FurD. melanogaster IAP1-BIR1 (PDB: 3SIP), a representative structure of a canonical BIR domain, with an RMSD of 2.4 Å , αAB′ 232–242), αB′ (247–255), αC′ (277–281), and αD′ (297–309)) and one antiparallel β-sheet composed of three β-strands (β1′ (257–259), β2′ (265–268), and β3′ (272–276). The topology of the ule Fig. . The add2, αB′ Ω Fig. , but the205 Fig. e. Thus, D. melanogaster IAP1-BIR1  51 and H. sapiens nuclear-interacting partner of ALK 53 as ScPml39 structural orthologs. Both SpRsm1 and HsNIPA/ZC3HC1 feature a domain organization with two tandem zf-C3HC/Rsm1 modules and meet the criteria for conservation of ScPml39 residues essential for structural integrity  following Cys429 is also conserved in its putative Rsm1 module. Therefore, the NIPA/ZC3HC1 structure is expected to be highly similar to that of ScPml39 . Moreover, the additional helix αAB′ insertion is solely found in the Rsm1 module. These findings together with the difficulty to identify homologs on the sequence level suggest that the Pml39 fold has rapidly evolved, as only key residues are conserved. A possible sequence of events for the evolution of the Pml39 fold is as follows: (1) mutation in the CCHC zinc finger motif of an ancestral BIR domain, (2) domain duplication, (3) insertion of helix αAB′ and extra residues between helix αC and the zinc-coordinating histidine in the Rsm1 module. Steps of insertion/deletion are expected to make it more difficult for sequence algorithms to predict the Pml39 fold53. Indeed, the N-terminal consensus motif of canonical BIR domains, Rxx(S/T)Ω, could not be clearly identified in the Rsm1 module in silico  share conserved key residues responsible for the structural domain integrity, the ZnF motif in the zf-C3HC and Rsm1 families with the consensus sequence C-xico Fig. .ScPml39 has enabled us to unambiguously identify S. pombe SpRsm1 and human NIPA/ZC3HC1 as structural orthologs of ScPml39  and a DNA fragment encoding a C-terminally truncated (residues 1–325) construct of Saccharomyces cerevisiae Mlp1 (UniProtKB: Q02455) were amplified by PCR from genomic DNA and cloned into the NcoI/NotI restriction sites of the pET28a vector (Novagen). The constructs were overexpressed in E. coli BL21(DE3)-RIL CodonPlus cells (Agilent Technologies) and grown in LB medium containing appropriate antibiotics. Protein expression was induced at OD600 of ~ 0.6 with 0.1 mM isopropyl-β-d-thiogalactoside (IPTG) at 18 °C for 16 h. The cells were harvested by centrifugation at 7500×g and 4 °C and lysed with a cell disruptor (Avestin) in a buffer containing 20 mM Tris–HCl, pH 8.0, 300 mM NaCl, 14.3 mM β-mercaptoethanol, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) (Sigma), 2 µM bovine lung aprotinin (Sigma), and complete EDTA-free protease inhibitor cocktail (Roche). After centrifugation at 35,000×g for 45 min, the cleared lysate was loaded onto a Ni-NTA column (Qiagen) and eluted with an imidazole gradient. Protein-containing fractions were pooled, dialyzed against a buffer containing 20 mM Tris–HCl, pH 8.0, 5 mM dithiothreitol (DTT), and 250 mM NaCl for full-length ScPml39, 100 mM NaCl for ScPml3977–317, or 150 mM NaCl for ScMlp11–325, and subjected to cleavage with PreScission protease  for 5 h at 4 °C. Following hexahistidine-tag removal, ScPml39 proteins were bound to a HiTrap SP column  and eluted with a NaCl gradient. For ΔC-ScMlp1, a HiTrap Q column was used. Protein-containing fractions were pooled, concentrated, and purified on a HiLoad Superdex 200 (16/60) gel filtration column  in a buffer containing 20 mM HEPES–NaOH, pH 7.5, 200 mM NaCl, and 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Protein concentrations were measured by absorbance at 280 nm, the proteins were flash-frozen in liquid nitrogen and stored at − 80 °C.DNA fragments encoding full-length (residues 1–334) and truncated (residues 77–317) ScPml39 at 1.3 mg/ml was incubated with a dilution series of 2 mg/ml porcine elastase at room temperature for 30 min. An aliquot of each dilution was mixed with reducing SDS-PAGE sample buffer and analyzed by SDS-PAGE. The remaining reaction volumes were quenched by guanidinium chloride powder for mass spectrometry analysis. To this end, the samples were run over a reversed phase column (PLRP-S), collected peaks were injected into an ion trap mass spectrometer, and spectra were analyzed by GPMAW58.In a volume of 100 µl, full-length ScPml3977–317 were grown at 20 °C in hanging drops containing 1 μl of the protein at 10 mg/ml and 1 μl of a reservoir solution consisting of 12% (w/v) PEG 8,000 and 0.1 M HEPES–NaOH, pH 7.7. Crystals grew in space group P3121 within a week, were cryo-protected in 25% (v/v) glycerol containing 12% (w/v) PEG 8000, and 0.1 M HEPES–NaOH, pH 7.7, and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at the beamlines X29A at the National Synchrotron Light Source (NSLS) of the Brookhaven National Laboratory (BNL) and 8.2.2 at the Advanced Light Source (ALS). Diffraction data were processed in HKL200059. The structure was solved by the single anomalous dispersion (SAD) phasing technique running the script AutoSol of the PHENIX package60. The asymmetric unit contained one molecule. Model building was performed in O61 and Coot62. The final model spanning residues 79–311 was refined in PHENIX to Rfree/Rwork factors of 26.3%/23.3% with excellent stereochemistry and clash score as assessed by MolProbity63. Details for data collection and refinement statistics are summarized in Table 64. Atomic coordinates and structure factors have been deposited with the Protein Data Bank under the accession code 7RDN.Crystals of ScPml3977–317 was injected into 350 µl of 70 µM ScMlp11–325 in the chamber every 180 s. Baseline-corrected data were analyzed using the ORIGIN software to determine the molar ratio (n), dissociation constant (KD), and enthalpy (ΔH). These parameters were subsequently used to determine the free Gibbs energy (ΔG) and the entropic component (TΔS) using ΔG = − RT ln (1/KD) and TΔS = ΔH − ΔG equations, where R and T are the gas constant ) and absolute temperature, respectively. Thermodynamic parameters are represented as mean values ± standard deviation calculated from three independent measurements.ITC measurements were performed at 4 °C using a MicroCal auto-iTC200 calorimeter . Samples were extensively dialyzed against a buffer containing 500 mM NaCl, 20 mM HEPES (pH 7.5), and 0.5 mM TCEP. After dialysis, the protein was filtered (0.22 µm) and centrifuged, followed by determining their concentration by UV absorbance at 280 nm. 2 µl of 1.6 mM ScMlp11–325 with a Superdex 200 (10/300) GL column  at 0.5 mL min−1 at 25 °C in 500 mM NaCl, 20 mM HEPES–NaOH (pH 7.5), and 0.5 mM TCEP. Absolute molecular weights were determined using MALS. The scattered light intensity of the column eluent was recorded at 18 different angles using a DAWN-HELEOS MALS detector (Wyatt Technology Corp.) operating at 658 nm after calibration with the monomer fraction of Type V BSA (Sigma). Protein concentration of the eluent was determined using an in-line Optilab T-rex interferometric refractometer (Wyatt Technology Corp.). The weight-averaged molecular weight of species within defined chromatographic peaks was calculated using the ASTRA software version 6.0 (Wyatt Technology Corp.), by construction of Debye plots (KC/Rθ versus sin2[θ/2]) at 1-s data intervals. The weight-averaged molecular weight was then calculated at each point of the chromatographic trace from the Debye plot intercept, and an overall average molecular weight was calculated by averaging across the peak.SEC experiments were performed on 100 µl injections of 70 µM S. cerevisiae strains used in this study -KanMX as templates65. Expression of the three transgenes is driven by the PML39 endogenous promoter (300 bp upstream the ATG codon). Unless indicated, cells were grown at 30 °C in rich  or Synthetic Complete (SC) media55 and harvested during exponential phase.All dy Table  are haplScPml39 or SpRsm1 were acquired using a Leica DM6000B microscope with a 100 ×/1.4 NA (HCX Plan-Apo) oil immersion objective and a CCD camera , and further scaled equivalently using the MetaMorph software (Molecular Devices). Whole-cell extracts were prepared from cells grown in SC and analyzed by SDS-PAGE using stain-free precast gels (Biorad) followed by western-blotting with monoclonal anti-GFP antibodies 66. Growth assays were achieved by spotting serial dilutions of cells on SC medium and incubating the plates at 25 °C.Localization of tagged fluorescent proteins was analyzed in live cells grown in SC medium. Wide-field fluorescence images of GFP-tagged versions of Supplementary Information.


The molecule of the title compound is almost planar but with a dihedral angle between the two phenyl rings of 19.22 (5)°. In the crystal, molecules are linked by C—H⋯O interactions, forming sheets in the °. The molecules are linked by C—H⋯O interactions, forming sheets in the (21The structure of the title compound, C They contain an α-β unsaturated carbonyl system, which is present in open-chain form, and two aromatic rings are joined through three-carbon atoms -1-(4-methylphenyl)-3-(4-nitrophenyl)prop-2-en-1-one -1-(3-bromophenyl)-3-prop-2-en-1-one -3-(3-nitrophenyl)-1-[4-(piperidin-1-yl)phenyl]prop-2-en-1-one -1-[4-(prop-2-ynyloxy)phenyl]prop-2-en-1-one Fig. 1, which c. There are no π–π interactions between the phenyl rings.The molecules are linked by C–H⋯O interactions Table 1, which fm-nitro benzaldehyde  was added. The resulting mixture was stirred at room temperature for 30 min. Then, the separated solid from the reaction mixture was filtered, washed with cold water, dried and recrystallized from ethanol:dimethylformamide mixture (9:1). Golden yellow crystals . The reaction scheme is shown in Fig. 3−1 (KBr): 2987 , 2117 (C≡C str), 1650 (C=O), 1518 (asym NO2 stretch),1444 (sym NO2 stretch), 1252 (C—O stretch); 1H NMR : 7.55 , 7.36 , 7.28 , 7.16 , 7.09 , 6.73 , 4.46 , 2.79 .A well-stirred solution of 1-[4-(prop-2-ynyloxy)phenyl]ethanone  in 20 ml of ethanol was added slowly to alcoholic potassium hydroxide . To this solution, Crystal data, data collection and structure refinement details for the title compound are summarized in Table 210.1107/S2414314622009579/bt4126sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314622009579/bt4126Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314622009579/bt4126Isup3.cmlSupporting information file. DOI: 2210152CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


T, was isolated from the sediment collected from Daya Bay, Guangdong, PR China. Optimal growth occurred at 37–40 °C, pH 6.0 and in the presence of 4 % NaCl. The 16S rRNA gene sequences analysis revealed that DP3N28-2T showed highest similarities with Mameliella alba DSM 23384T (98.3 %), Antarctobacter jejuensis 13–2-B6T (97.2 %), Antarctobacter heliothermus El-219T (96.8 %), Maliponia aquimaris MM-10T (96.7 %), Ponticoccus litoralis CL-GR66T (96.4 %) and Aquicoccus porphyridii L1 8-17T (96.1 %). The predominant fatty acids (>10 %) were summed feature 8  and C16 : 0 (11.0 %). The polar lipids contain phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, one aminophosphlipid, one phospholipid and three unidentified lipids. The respiratory quinone was Q-10. The DNA G+C content was 63.0 mol% (data from the genome sequence). The estimated genome size was 5.12 Mb. The average nucleotide identity values between the DP3N28-2T genome and the genome of M. alba was 81.1 %, while the digital DNA–DNA hybridization value was 23.4 %. The phenotypic, genotypic and chemotaxonomic differences between DP3N28-2T and its phylogenetic relatives indicates that DP3N28-2T should be regarded as representing a novel species of the genus Mameliella, for which the name Mameliella sediminis sp. nov. is proposed. The type strain is DP3N28-2T (=MCCC 1K06218T=KCTC 82804T).A Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacterium, capable of producing poly-β-hydroxyalkanoate, designated DP3N28-2 Mameliella was proposed by Zheng et al. in 2010, with Mameliella alba as the type species [Rhodobacteraceae was validly published as the family Roseobacteraceae [Mameliella was moved to the family Roseobacteraceae of the class Alphaproteobacteria. In 2015, four heterotypic synonyms of Mameliella alba, Mameliella phaeodactyli [Mameliella atlantica [Ponticoccus lacteus [Alkalimicrobium pacificum [T (EU734592.1) had been submitted to the GenBank database [Mameliella contains only one species with a correct validly published name according to List of Prokaryotic names with Standing in Nomenclature (https://lpsn.dsmz.de/genus/mameliella).The genus raceae , therefoactyli , Mamelacteus  and Alificum , have bedatabase . After iMameliella, designated DP3N28-2T. Using PCR with specific primers [T had a poly-β-hydroxyalkanoate (PHA) polymerase (phaC) gene (gel profile data not shown), which indicated the potential for producing PHA. Therefore, in this study, we were aimed to determine the exact taxonomic position of DP3N28-2T, by using a polyphasic approach based on phenotypic, chemotaxonomic and genotypic data, and estimated the PHA-producing ability of the genus Mameliella.During a course of study on the poly-β-hydroxyalkanoate-producing marine bacteria from Daya Bay, Guangdong, PR China, we isolated a novel bacterial strain resembling members of the genus  primers , we idenMameliella was isolated from a surface sediment sample near off-shore sewage outfall  from 5.3 m depth. The sediment sample was incubated in medium containing 5 g (w/w) peptone and 1 g (w/w) yeast extract per litre sea water, at 30 °C for 28 days, and then isolated using the standard dilution plating technique on marine agar . After purification by re-streaking, the novel strain was routinely cultivated at 30 °C on MA and stored at −80 °C in sterile 15 % (v/v) glycerol supplemented with 1 % (w/v) saline. The reference type strain, M. alba CGMCC 1.7209T, were procured from the China General Microbiological Culture Collection Centre. Both strains could form single colonies on MA within 24 h at 30 °C. To standardize the culture conditions in this study, we cultivated M. alba CGMCC 1.7209T using MA or MB.The novel isolate of a member of the genus T was amplified by PCR with the universal primers 27F and 1492R [T was analysed by using the blast programme (NCBI) and compared with closely related sequences of reference organisms using the service from EzBioCloud (www.ezbiocloud. net) [clustal_x, version 1.81 [mega 7 programme package [T obtained by amplification (1303 bp) was included in the 16S rRNA gene sequence assembled from genomic sequences (1437 bp). On the basis of the 16S rRNA gene sequence (from genome sequence), strain DP3N28-2T was closely related to Mameliella alba DSM 23384T (98.3%), Antarctobacter jejuensis 13–2-B6T (97.2%), Antarctobacter heliothermus El-219T (96.8%), Maliponia aquimaris MM-10T (96.7%), Ponticoccus litoralis CL-GR66T (96.4%) and Aquicoccus porphyridii L1 8-17T (96.1%). In the NJ, MP and ML phylogenetic trees  Genome Server provided by DSMZ [T formed a clade with the type strain of M. alba  . Alignmeion 1.81 , and posion 1.81 , maximumion 1.81  methods  package . The MP ic trees , althoug by DSMZ  to furth. alba , which iT was sequenced using a HiSeq X-ten platform (Illumina), assembled at Guangdong Magigene Biotechnology.  using the SPAdes software (v3.13.0) [T (JAHUZG000000000) had a total length of 5 120 735 bp, coverage of 600×, contig N50 value of 1 507 360 bp, 4966 predicted gene sequences and 54 RNAs. The final assembly contained 21 contigs and a had a DNA G+C content of 63.0 mol%.The draft genome of DP3N28-2v3.13.0)  and annov3.13.0) . The assT and the type species M. alba CGMCC 1.7290T (FMZI01000000). ANI and dDDH (formula 2) were 81.1 and 23.4 %, respectively. They were both lower than the threshold value for species demarcation . Furthermore, the difference in DNA G+C content between DP3N28-2T and the type species was 2.2 mol%, which indicated that they represented two different species [T was considered to represent a novel species of the genus Mameliella.Average nucleotide identity (ANI) using the OrthoANI algorithm  and digiT were examined after incubation in MB or on MA at 30 °C for 24 h to a maximum of 1 week unless otherwise specified. Cell morphology, Gram-staining and motility were determined by microscopy  and transmission electron microscopy . Cells of DP3N28-2T were Gram-stain-negative non-flagellated and rod shaped. The cell length was 1.3–4.7 µm and the width was 0.5–0.9 µm . The shape varied due to the distribution of the PHA granules. Growth of DP3N28-2T and M. alba CGMCC 1.7290T was tested at 4, 8, 15, 20, 25, 30, 35, 37, 40, 45 and 50 °C, NaCl concentrations in the range of 0–10 %  and pH values in the range of 4.0–12.0  adjusted using HCl or NaOH. The growth was measured using OD600 after 24, 48 and 96 h. Growth occurred at temperatures from 15 to 45 °C , pH from 5.0 to 8.0  and NaCl concentrations from 0–10 % . No growth observed after incubation in an anaerobic chamber on MA for 2 weeks. Oxidase activity was determined using a bioMérieux oxidase reagent kit according to the manufacturer’s instructions. Catalase activity was tested by bubble production using 3 % (v/v) H2O2 solution. Utilization of citrate, pyruvate and malonate as sole carbon sources was determined. DP3N28-2T was cultivated in the minimal salt medium containing, per litre of distilled water: 2.28 g K2HPO4·3H2O, 1.32 g (NH4)2SO4, 0.47 g NaHPO4, supplemented with 1 % carbon source. Hydrolysis of agar and starch, H2S production, reduction of nitrate, Voges–Proskauer test, methyl red test and indole production were investigated according to the methods described by Dong and Cai [T and the reference type strain independently twice without replicates at 30 °C. The polyhydroxyalkanoates production of DP3N28-2T was examined as described by Juengert et al. [All morphological and physiological features of DP3N28-2T exhibited phenotypic similarities to M. alba CGMCC 1.7290T, including being Gram-stain-negative, rod shaped, aerobic, having positive reactions for catalase and oxidase and identical reactions in API ZYM tests, except for α-galactosidase. Both strains could accumulate poly-β-hydroxybutyrate. The GC result indicated that DP3N28-2T and M. alba CGMCC 1.7290T accumulated PHA up to 12.3±2.9 % and 19.2±5.8 % , respectively. In the genomic data, the PHA synthase gene (phaC), PHA depolymerase gene (phaZ), regulator gene (phaR) and PHA granule-associated phasin gene (phaP) were annotated (accession numbers are listed in Table S1).DP3N28-2T from M. alba in various ways  as the critical phenotypic differences between DP3N28-2T and M. alba. The detailed physiological and biochemical characteristics of DP3N28-2T are given in T was susceptible to troleandomycin, nalidixic acid, aztreonam and niaproof 4, and resistant to lincomycin, vancomycin, fusidic acid, rifamycin SV, guanidine HCl and minocycline. Cultures of DP3N28-2T and M. alba CGMCC 1.7290T were grown in MB at 30 °C for 48 h, and then freeze-dried for polar lipid, respiratory quinone and cellular fatty acid composition analysis. The cellular fatty acid composition was measured by the Marine Culture Collection of China  using the Microbial ID system [T was determined by the MCCC using ultra-performance liquid chromatography–tandem mass spectrometry methods [16 : 0 and C18 : 0 as the top three components (>5 %), however, in M. alba CGMCC 1.7290T, the proportion of C18 : 0 was higher than that of C16 : 0, which were consistently shown in all the five representatives of M. alba [T included phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, one aminophospholipid, one phospholipid and three unidentified lipids. The polar lipid composition was variable among the M. alba strains [, but none of them contained phosphatidylmonomethylethanolamine, which could be used to distinguish DP3N28-2T from M. alba.On the basis of the data obtained during this study, we could distinguish DP3N28-2ous ways . However was 3.5 , from whD system . Respira methods . The cel methods . Total l methods . The resT as representing a novel species of the genus Mameliella, for which the name Mameliella sediminis sp. nov. is proposed.On the basis of these phenotypic, chemotaxonomic, phylogenetic and genetic data, we propose to classify strain DP3N28-2Mameliella sediminis .2S production. Pyruvate are used as sole carbon source, but not citrate. In the API ZYM tests, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-galactosidase; negative for trypsin, α-chymotrypsin, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. In the API 20NE tests, positive for d-glucose fermentation, urease, β-glucosidase (aesculin hydrolysis) and β-galactosidase; weakly positive for gelatin hydrolysis, utilization of d-glucose, l-arabinose, and d-mannose; negative for reduction of nitrate to nitrite, denitrification, indole production, arginine dihydrolase, utilization of d-mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, capric acid, malic acid, adipic acid, phenylacetic acid and trisodium citrate. According to Biolog GEN III MicroPlate assays, α-hydroxybutyric acid, l-arginine, l-lactic acid, β-hydroxy-d,l-butyric acid, d-fucose, acetoacetic acid and trehalose are oxidized. The polar lipids are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, one aminophosphlipid, one phospholipid and three unidentified lipids. The main fatty acids are summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) and C16 : 0. The respiratory quinone is Q-10.Cells are Gram-stain-negative, aerobic, and non-motile rods (0.5–0.9 µm wide and 1.3–4.7 µm long). Small colonies (1–2 mm) are produced on MA after incubation at 30 °C for 24 h. Colonies are smooth, convex and beige. Growth occurs at 15–45 °C, at pH 5.0–8.0 and in the presence of 0–10 % NaCl; optimal growth occurs at 37–40 °C, at pH 6.0 and in the presence of 4.0 % NaCl. Positive for catalase and oxidase. Negative for nitrate reduction, hydrolysis of gelatin and starch, methyl red test, Voges–Proskauer reaction, and HT (=MCCC 1K06218T=KCTC 82804T) was isolated from the sediment collected from Daya Bay, Guangdong, PR China. The DNA G+C content of the type strain is 63.0 mol % (data from genome sequence). The GenBank accession number for the 16S rRNA gene sequence and draft genome of strain DP3N28-2T are MZ277401 and JAHUZG000000000 respectively.The type strain, DP3N28-2Click here for additional data file.


It has a slightly lower Al content than the closely related η-Al11Cr2 phase.Al J. Inst. Met. 60, 319–337] as having the composition Al11Cr2 (Al:Cr ratio = 5.5:1). Its crystal structure was later refined  to have a slightly lower Al content (Al:Cr ratio = 5.16:1). In the present work, a monoclinic phase with composition Al2.95Cr0.59 (Al:Cr ratio = 5.04:1) was obtained by high-pressure sintering (HPS) of a stoichiometric Al11Cr2 mixture. Structure analysis of this phase, hereafter named η′, revealed a close relationship to the previously reported η-Al11Cr2 structure, but with different mixed-occupied sites. Five fully occupied sites exhibit refined site occupation factors of 0.899 (5), 0.984 (4), 0.977 (5), 0.946 (4) and 0.945 (4) for the corresponding Al atoms. Moreover, there are no split Al sites in the η′-structure as reported for the η-structure. The refined chemical composition of the η′-phase revealed that it comprises two Al atoms fewer and two Cr atoms more than the previously reported η-Al11Cr2 phase.The monoclinic η-phase in the binary Al–Cr system was initially named by Bradley & Lu [Bradley & Lu 1937. J. Inst Icosahedral quasicrystals from the Al–Cr alloy containing 7 to 15 at.% Cr have twinned Al7Cr as final decomposition product while the equilibrium η-Al11Cr2 phase is completely absent during the decomposition of quasicrystals . Therefore, it is pivotal to decipher the formation of the Al11Cr2 phase in order to enhance our understanding of the formation of quasicrystals as well as precipitations in the Al–Cr binary system. For the present investigation, we used high-pressure sintering (HPS) of a stoichiometric Al:Cr mixture (molar ratio = 11:2) for crystal growth.The Al-rich part of the binary Al–Cr system has been investigated intensively in the past. One of the reported phases is monoclinic η-Al& Lu 1937. Its cryLu 1937a to have Lu 1937a, but lat al. 1986 shortly  al. 1987 found th al. 1988 also fou11Cr2. Its crystal structure is closely related to the η-Al11Cr2 phase previously reported by Cao & Kuo , viz. Al4/Cr15, Al5/Cr16, Al11/Cr17, Al13/Cr18 and Al15/Cr19 with refined site occupation factors (s.o.f.) of 0.899 (5), 0.984 (4), 0.977 (5), 0.946 (4) and 0.945 (4) for Al4, Al5, Al11, Al13 and Al15, respectively. Quite differently, in the η-Al11Cr2 model there are six Al/Cr mixed-occupied sites (with an s.o.f. of 0.8291 for Al in three sites and 0.8291 for Cr in the remaining three sites) and 15 disordered positions for Al atoms, all split into two sites with a ratio of 0.8291:0.1709. A detailed comparison of coordinates and occupancies for related atoms in the crystal structure models of η′-Al11Cr2 and η-Al11Cr2 can be found in the supporting information (Table S1).We have named the present phase η′-Aluo 2008a, however11Cr2 was described in detail  micrographs and energy dispersive X-ray spectroscopy (EDS) analysis of a fragment from which single crystals were selected for X-ray diffraction studies revealed that the η- and η′-Al11Cr2 phases have a very similar (and based on this method indistinguishable) chemical composition . However, the refined chemical composition of the present η′-Al11Cr2 phase using single-crystal X-ray analysis reveals that it has two Al atoms fewer and two Cr atoms more than the reported η-Al11Cr2 phase.Since the crystal structure of η-Alet al., 2018The high-purity elements Al  and Cr  were mixed in the stoichiometric ratio 11:2 and ground in an agate mortar. The blended powders were placed into a cemented carbide grinding mound of 9.6 mm diameter and pressed at 4 MPa for about 5 min. A uniformly cylindrical block with 9.6 mm in diameter and 10.0 mm in height was obtained that was subsequently loaded into a six-anvil high-temperature high-pressure apparatus as described elsewhere  I. DOI: 10.1107/S2414314620014121/wm4139Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2414314620014121/wm4139sup3.pdfSupplementary material . DOI: 2040163CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N,N-di-n-propyltryptamine (4-HO-DPT) is reported.The first single-crystal structure of the hydrochloride salt of a psilocin analogue, specifically 4-hydroxy- N,N-di-n-propyltryptammonium (4-HO-DPT) chloride {systematic name: N-[2-(4-hydroxy-1H-indol-3-yl)ethyl]-N-propylpropan-1-aminium chloride}, C16H25N2O+·Cl−, has a singly protonated tryptammonium cation and one chloride anion in the asymmetric unit. A series of N—H⋯Cl and O—H⋯Cl hydrogen bonds connect the ions together in ladder chains along [010].The title compound, 4-hydroxy- N,N-dimethyl-4-phosphoryloxytryptamine, is a naturally occurring tryptamine found in ‘magic mushrooms’. When consumed orally, the phosphate group of psilocybin is hydrolyzed to generate psilocin, 4-hydroxy-N,N-dimethyltryptamine. Psilocybin functions as a prodrug of psilocin, its active metabolite, which binds to and stimulates the human serotonin 2a receptor  have shown incredible potential for treating a wide variety of the world’s most debilitating, intractable, and costly health problems, including anxiety, depression, addiction, post-traumatic stress disorder (PTSD) and inflammation  is a structural analogue of psilocin and produces similar  psychedelic effects in human subjects  chloride °. The N—H of the indole ring, and the N—H of the ammonium group also both hydrogen bond to other symmetry-generated chloride anions through N—H⋯Cl interactions  was dissolved in dichloromethane, and 160 µL of hydorochloric acid  were added with stirring at room temperature. The mixture was stirred for 30 minutes, resulting in a white precipitate which was isolated via vacuum filtration and washed with diethyl ether to yield 28 mg of the salt. A second crop was collected by concentrating and cooling the filtrate to give another 13 mg of the salt (73% yield). Crystals suitable for X-ray diffraction studies were grown by the slow evaporation of a methylene chloride/methanol mixture. The sample was analyzed by nuclear magnetic resonance. 1H NMR : δ 7.07 , 7.04–6.96 , 6.47 , 3.42–3.30 , 3.20–3.07 , 3.00 , 1.60 , 0.84 ; 13C NMR : δ 149.9 (ArC), 138.8 (ArC), 123.3 (ArC), 123.2 (ArC), 115.9 (ArC), 108.5 (ArC), 104.5 (ArC), 103.7 (ArC), 54.7 (AkC), 53.7 (AkC), 21.2 (AkC), 16.8 (AkC), 10.0 (AkC).Freebase 4-hydroxy-Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620015461/sj4218sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314620015461/sj4218Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620015461/sj4218Isup3.cmlSupporting information file. DOI: 2045782CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The thermodynamic study showed that the activation energy (Ea) was 70.37 kJ·mol−1, the activation enthalpy change of the reaction (ΔH) > 0, the free energy of activation (ΔG) > 0 and the activation entropy change (ΔS) < 0, indicating the reaction could not be spontaneous. The reaction mechanism of ZEN was studied by a hybrid quadrupole orbitrap mass spectrometer. It was found that ZEN first generated the intermediate G/L/D/W-ZEN+H2O, followed by generating the intermediate W-ZEN-H2O under the action of a degrading enzyme. Then, the lactone bond was opened to produce C18H24O6, and finally the decarboxylation product C17H24O4 formed automatically. The kinetics and thermodynamics of the enzymatic degradation of zearalenone (ZEN) in degummed corn oil were investigated by analyzing the impacts of temperature, pH, ZEN hydrolase dosage and ZEN concentration on the initial reaction rate. The kinetic study found that the maximum reaction rate was 0.97 μmol × kg Fusarium roseum, Fusarium graminearum, Fusarium tricinum and other Fusarium microorganisms during their secondary metabolism and is the most widely contaminated mycotoxin in the world /.According to the Lineweaver Burk method, the intersection point with the Y axis is Vm−12, Vm0 = (βε − δ2)/Δ = 0.9538 μmol × kg−1 × min−1, Km0 = (βγ − αδ)/Δ = 11,052 μmol × kg−1. Vm0 and Km0 are the estimated values of the maximum reaction rate and Michaelis constant, respectively.The Wilkinson statistical method includes two steps of nonlinear multiplication and Taylor expansion as shown in The f and f` in −12, b1 = (β`δ`−γ`ε`)/Δ` = 1.01698, b2 = (α`ε`−γ`δ`)/Δ` = 434.9822, so that Vmax = Vm0 × b1 = 0.97 μmol × kg−1 × min−1, Km = Km0 + b2/b1 = 11,476 μmol × kg−1.According to The final Michaelis equation calculated by the above statistical method is V = 0.97[S]/.There is a certain difference between Vm and Km calculated by the Lineweaver buck method and the Wilkinson statistical method. The Wilkinson statistical method is considered to be reliable . Therefomax obtained in this study and the data reported by other scholars + parent ion (m/z 319.15) and [C18H22O5-H2O]+ fragment ion (m/z 301.14) under mass spectrometry conditions. Francesca et al. [It can be seen from a et al.  reportedThe chromatogram of the negative ion scanning results of the enzymolysis reaction products is shown in 18H24O6-H]− (m/z 335.15) and [C18H22O5-H]− (m/z 291.16) mother ions, respectively.The mass spectra of B and C in It can be seen from The chromatogram of the positive ion scanning results of the enzymolysis reaction products is shown in m/z 808.61. Further, the difference is calculated by the fragment ions  to get exactly three amino acid residues, namely glycine , leucine  and tryptophan . The above amino acid residues correspond to neutral losses Δ57, Δ113 and Δ186, respectively. Further calculation of the molecular weight shows that the substance also contains aspartic acid residues . As aspartic acid belongs to an acidic amino acid, it is easy to lose a water molecule (H2O) and add an additional positive charge under the condition of collision-induced dissociation (CID), so the whole molecular system shows a double charge . Finally, based on the research results of Qi et al. /.(2) The kinetic study found that the maximum reaction rate was 0.97 μmol × kg(3) Thermodynamic study shows that the activation energy (Ea) is 70.37 kJ·mol, the activation enthalpy change (ΔH) > 0, the free energy (ΔG) > 0, the activation entropy change (ΔS) < 0 and the reaction cannot be spontaneous.2O, and then produced the intermediate W-ZEN-H2O under the action of the degrading enzyme. Then, the lactone bond was opened to produce the water-added product C18H24O6, and finally the decarboxylation product C17H24O4 formed automatically. (4) The reaction mechanism of ZEN was studied by liquid chromatography mass spectrometry. It was found that ZEN first produced the intermediate G/L/D/W-ZEN+H−1. Zearalenone hydrolase was obtained from Rongvezyme Preparation Co., Ltd., Zhuhai, China; the enzyme activity was 243 U × g−1, which was measured according to the methods of Wang  was used at a flow rate of 1 mL × min−1.Separation was conducted by a CDegummed corn oil (500 g), 0.1 mol/L Tris HCI buffer  and 15 mg ZEN hydrolase were added to a beaker. Then, the sample was added to a high-speed shear machine  to mix the samples at 5000 r/min for 2 min. After that, the mixed sample (100 g) was put into the enzyme reactor at a certain temperature. After 10 min, the temperature was raised to 95 °C to terminate the reaction. Then, the sample was separated by a centrifuge at 10,000 r/min for 10 min. The upper oil layer was dried at 0.09 mPa and 80 °C. Finally, the content of the ZEN of the sample was determined by HPLC.−1 × min−1); M0 is the content of ZEN in DCO after the reaction (μg × kg−1); M1 is the content of ZEN in DCO before the reaction (μg × kg−1); t is the reaction time (10 min); M is the molar mass of ZEN (318 g × mol−1).The initial reaction rate was calculated by the equationTo study the effects of pH on the initial reaction rate of ZEN degradation, different buffers were used for pH 6.5–9.0.−1.To study the effect of ZHD dosage on the initial reaction rate of ZEN degradation, different ZHD dosages were used for 10–60 mg × kg−1. Lineweaver-Burk plot and Wilkinson statistical method were applied to calculate the Michaelis constant (Km) and maximum velocity (Vmax).To study the effect of the concentration of ZEN on the initial reaction rate of ZEN degradation, different concentrations of ZEN were used for 500–7000 μg × kg−1 × K−1); T, the absolute temperature in Kelvin.The effect of reaction temperature on the initial reaction rate of ZEN degradation was investigated at a range of temperatures between 25 and 50 °C. The activation energy (Ea) was calculated from the slope of the Arrhenius plot according to the equation: −34 J × s); Kb, Boltzmann’s constant (1.3807 × 10−23 J × K−1); T, Absolute temperature (K); N, Avogadro’s number (6.02 × 1023 mol−1); R, Gas constant (8.314 J × mol−1 × K−1)The thermodynamic parameters for substrate hydrolysis were calculated according to equation .kcat = Liquid chromatographic conditions: chromatographic column: Thermo Accucore RP-MS 100 mm × 2.1 mm, particle size 1.8 μm. Column temperature: 30 °C, injection volume: 5 μL. Flow rate: 0.3 mL/min. Mobile phase: positive ion mode: Water phase (A): 0.1% formic acid aqueous solution; organic phase (B): 0.1% formic acid acetonitrile. Negative ion mode: aqueous phase (A): 0.03% ammonia aqueous solution, organic phase (B): 0.03% ammonia acetonitrile. Gradient elution: positive and negative are the same. The gradient ratio of mobile phase is 0 min, 95% A, 5% B, 15 min, 5% A, 95% B, 17 min, 5% A, 95% B, 17.1 min, 95% A, 5% B, 20 min, 95% A, 5% B.m/z, Resolution: 70,000. Polarity: Positive, AGC target: 1 × 106, Max inject time: 50. Positive ion mode ion source parameters: Sheath gas flow rate: 40, Aux gas flow rate: 5, Sweep gas flow rate: 1, Spray voltage (KV): 3.2, Capillary temperature (°C): 320, S-lens RF level: 55.0. Aux gas heater temperature (°C): 400. Anion scanning parameters: Scan type: Full MS, Scan range: 70.0 to 1050.0 m/z, Resolution: 70,000, Polarity: Negative, AGC target: 1 × 106, Max inject time: 50. Anion mode ion source parameters: Sheath gas flow rate: 40, Aux gas flow rate: 5, Sweep gas flow rate: 1, Spray voltage (KV): 2.8, Capillary temp. (°C): 320, S-lens RF level: 55.0, Aux gas heater temp. (°C): 400.Mass spectrum conditions: Positive ion scanning parameters: Scan type: Full MS, Scan range: 70.0 to 1050.0 All experiments were carried out in triplicate independently and SPSS  was used for data analysis. Results are presented as mean ± standard deviation of triplicate measurements.