Low moisture content, flavonoids, phenols, antioxidants, fatty acid (FA) compositions, high PV, and poor sensory quality—i.e., excessive browning, an exceptionally crunchy texture, and a bitter flavor—were all characteristics of kernels roasted at 150 °C. With a perfect crispy texture, a rich brown color, and a strong nutty flavor, kernels roasted at 125 °C had lower PV; higher oxidation-resistant UFA compositions; considerable concentrations of flavonoids, phenols, and antioxidants; and good sensory quality. Therefore, ‘A4′ and ‘Beaumont’ kernels could be roasted at 125 °C for use in the industry to improve kernel quality and palatability.Depending on the temperature regime used during roasting, the biochemical and sensory characteristics of macadamia nuts can change. ‘A4′ and ‘Beaumont’ were used as model cultivars to examine how roasting temperatures affected the chemical and sensory quality of macadamia nuts. Using a hot air oven dryer, macadamia kernels were roasted at 50, 75, 100, 125, and 150 °C for 15 min. The quantity of phenols, flavonoids, and antioxidants in kernels roasted at 50, 75, and 100 °C was significant ( Macadamia integrifolia) is the top-ranking nut in the world and is frequently consumed either roasted or as an ingredient in different confectionery items. The widespread intake of macadamia nuts may be attributed to its high nutritional value and alleged health benefits, which include reducing the risk of type 2 diabetes and cardiovascular disease [According to ,2, macad disease ,4,5. The disease ,7,8,9. T disease . Macadam disease ,12.According to ,14, roasThe Maillard process occurs during roasting and produces a variety of volatile chemicals that give kernels their flavor and aroma ,20. AccoThe sensory attributes, nutritional value, and lipid quality of nuts can all be impacted by roasting, despite the fact that roasting has numerous positive effects . FurtherMacadamia integrifolia cultivars, were taken from Elliot Farm’s commercial orchards in Port Shepstone, KwaZulu-Natal, South Africa . According to standard industry procedure, nuts were harvested during the early season (May 2017) and the late season (June 2017). For further processing, approximately 20 kg of nuts from each cultivar were gathered on each sample day and brought to the Ukulinga Research Farm of the University of KwaZulu-Natal (UKZN).‘Beaumont’ and ‘A4′, two hybrids of Australian-bred db is moisture content on a dry mass basis, and At UKZN’s Ukulinga Research Farm, dehusking was carried out on the same day as harvesting using a 01-one-lane dehusker from WMC Sheet Metal Works in Tzaneen, South Africa. Nuts-in-shell (NIS) were dried using a mechanical convection oven  at the UKZN Postharvest Research Laboratory for three days in a row, starting at 35 °C on the first day, 38 °C for the second day, and 50 °C for the third day, according to a method described by  with mincracking .(2)KR=NThe roasting process was carried out in accordance with  with min® concentrator  while maintaining the sample’s oil content. The remaining oil was measured and reported using its dry weight.Using a Brabantia-table blender  Limited, Johannesburg, South Africa), kernels were ground into a fine powder after roasting. The amount of kernel oil was quantified from ground sample material using a method similar to that of  with a fv/v) was used to dissolve a 1 g sample of oil, and then a burette was used to add 1 mL of a saturated potassium iodide solution. Iodine was created after the reaction had occurred. Deionized water (50 mL) was added. Using a burette, iodine was titrated with Na2S2O3. The following formula was used to compute the peroxide value :2S2O3.With a few minor adjustments, the peroxide value (PV) was calculated in accordance with . In totaw/v) NaCl was added. To facilitate phase separation, the materials were agitated firmly and then centrifuged. The upper hexane phase (hexane containing FAMEs) was transferred to a gas chromatograph (GC) vial.With minor changes, the method described by ,34 was uGC  connected to a flame ionization detector (FID) was used to separate the FAMEs. A GC-FID system and a CTC Analytics PAL autosampler were connected. On a non-polar Stabil wax , FAs were separated. In this experiment, 1 mL/min of helium was employed as the carrier gas. At 240 °C, the injector temperature was maintained. A 5:1 split ratio was used to inject 1 L of the material. The oven’s temperature was set to 50 °C for 2 min, then increased to 180 °C at a rate of 25 °C per minute and held for 2 min, 200 °C at a rate of 3 °C per minute and held for 5 min, and lastly 240 °C at a rate of 4 °C per minute and kept for 15 min. Using the Chrom-Card data system version 2.3 software for Windows , each FA in the chromatogram was identified by comparing the retention times with certified standard mixes  and quantified by the peak areas on the chromatogram. In terms of g of FA per g of total FAs, the results were expressed. The total fatty acids (TFA), the ratio of saturated to unsaturated fatty acids (SFA/MUFA), and the ratio of monounsaturated to polyunsaturated fatty acids (MUFA/PUFA) were all calculated using the fatty acid composition that was obtained .v/v) methanol and vortexed for thirty seconds. The mixture was then agitated for an additional night at room temperature in order to remove the free phenols. Following centrifugation of the mixture, the supernatant was filtered through Whatman® no. 1 filter paper, and the sample was once again washed with 10 mL of solvent until the solvent was clear. The remaining kernel residue was then effectively released from cell wall-bound phenols using acid hydrolysis. Each sample received 10 mL of 60% aqueous methanol that had been acidified (2 M hydrochloric acid) before being submerged in a hot water bath for 90 min. Supernatants were filtered through a 0.45 m filter after being allowed to cool in glass tubes. Gallic acid monohydrate was used as a standard in the spectrophotometric determination of the total phenolic concentration, and the result was expressed as mg OF gallic acid equivalents (GAE) kg−1 of sample dry weight .Phenols were identified using  as a gui3) in accordance with an established procedure (kernel extract prepared for phenolic concentration determination). This procedure was based on that provided by [2 (0.03 mL) was added after that. It was let to stand at room temperature for 5 min. Then, 0.03 mL of 10% AlCl3 was added and allowed to react for 6 min before being mixed with 0.2 mL of 1 mM NaOH and diluted to 1 mL with purified water. At 510 nm, the absorbance of the reaction mixture was measured in comparison to methanol, which served as a blank. In terms of sample dry weight, the results were represented as mg quercetin kg−1.Using quercetin as a standard, the total flavonoid concentration was measured using aluminum chloride  free radical scavenging activity was assessed with some minor modifications from . First, −1 of sample dry weight.The 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) concentration was calculated using  with a fA randomized set of macadamia kernels were examined raw and immediately after roasting  18.1. Least significant difference values  were calculated for mean separation.The collected data were subjected to analysis of variance (ANOVA) using GenStat statistical software (GenStatp < 0.001) reduced as the roasting temperature rose , 75 °C (1.72 and 1.232 meq O2 kg−1), 100 °C (1.569 and 1.011 meq O2 kg−1), and 125 °C (1.161 and 0.947 meq O2 kg−1) significantly (p < 0.001) declined with increasing roasting temperatures. It is thought that kernels roasted at 125 °C have reduced PV, which is a sign of high quality. A high-quality macadamia product is defined by a low peroxide value of less than 1 meq O2 kg−1, which is deemed acceptable for a freshly refined fat and is categorized by a low oxidation state, according to [2 kg−1.From rding to ,46. Our rding to , who fou2 kg−1) and ‘Beaumont’ (1.682 meq O2 kg−1) saw an increase in PV after being roasted at 150 °C. This could be attributable to greater temperatures, which encourage the peroxide’s breakdown and polymerization into a variety of secondary compounds such as aldehydes and ketones [Both cultivars ‘A4′  and roasted kernels of ‘A4′ and ‘Beaumont’ macadamia nuts identified a total of thirteen individual FAs, among which oleic acid (C18:1n9c) and palmitoleic acid (C16:1) were predominant, followed by palmitic acid (C16:0), omega-6 (n-6), linoleic acid (C18:2n6c), stearic acid (C18:0), and cis-11-Eicosenoic acid (C20:1) . Additiop < 0.001) had the highest amounts of ∑ TFAs (1458.25 and 1165.55 µg/g), ∑ MUFAs (1250.10 and 959.68 µg/g), ∑ PUFAs (71.75 and 68.39), (∑ n-6)/(∑ n-3) (97.82 and 113.77 µg/g), linoleic acid (C18:2n6c) (64.34 and 52.70 µg/g), dihomo-γ-linolenic acid (C20:3n6) (5.26 and 15.22 µg/g), and omega-6 (n-6) (69.61 and 67.92 µg/g), respectively. The kernels are nutritious due to the large number of unsaturated fatty acids (UFAs), but they are also more prone to lipid peroxidation and rancidity, particularly when they have a high moisture content [p < 0.001) greater PV (2.432 and 1.741 meq O2 kg−1) and moisture content (1.5 and 1.46%), respectively. These values are within the acceptable range but indicate kernel susceptibility to the development of rancidity [p < 0.001) high ∑ SFAs (136.40 and 137.48 µg/g) and lower ∑ PUFA: ∑ SFA (0.43 and 0.38 µg/g), respectively. Our findings are similar to [ content . High ke content . This isancidity . Moreovemilar to .p < 0.001) reduced (p < 0.001) lower levels of linoleic acid (C18:2n6c) of 0.01 and 0.09 g/g, respectively. According to [2 kg−1 of the ‘A4′ and ‘Beaumont’ kernels, respectively, roasted at 150 °C had significantly (p < 0.001) higher PV, which is within the acceptable limit, but illustrates the vulnerability of kernels to the development of rancidity [With higher roasting temperatures, the fatty acid contents of ‘A4′ and ‘Beaumont’ kernels significantly  the largest concentrations of oleic acid (C18:1n9c) (751.97 and 624.92 g/g) and palmitoleic acid (C16:1) (472.96 and 340.36 g/g), respectively. This may be because some FAs are released when kernels are roasted at 125 °C, resulting in the synthesis of Maillard and caramelization browning products [p < 0.001) more oleic acid (C18:1n9c) (800.97 and 624.92 ug/g) and lower linoleic acid (C18:2n6c) (32.74 and 22.02 ug/g), respectively. Higher temperature during roasting can result to degradation of unsaturated fatty acids [Additionally, ‘A4’ and ‘Beaumont’ kernels roasted at 125 °C exhibited considerably  reduced PV , proving this. These PV show that the kernels are fresh and do not taste rancid because they are within the minimal permissible level (1–3 meq O2 kg−1) [Moreover, ‘A4’ and ‘Beaumont’ kernel roasted at 125 °C are less prone to lipid oxidation and the formation of rancidity. ‘A4’ and ‘Beaumont’ kernels roasted at 125 °C had significantly (O2 kg−1) ,48.−1), 75 °C (0.095 and 0.137 mg GAE kg−1), 100 °C (0.089 and 0.130 mg GAE kg−1), 125 °C (0.082 and 0.127 mg GAE kg−1), and 150 °C (0.038 and 0.087 mg GAE kg−1). The majority of phenolic chemicals, according to [Phenolic concentration was discovered to be inversely correlated with flavonoid concentration, with phenolic concentrations of ‘A4′ and ‘Beaumont’ roasted at 50 °C  . According to [With increasing roasting temperature, the flavonoid concentrations of ‘A4′ and ‘Beaumont’ kernels decreased significantly  decreased with rising roasting temperature. Compared to kernels roasted at a higher temperature of 150 °C , ‘A4′ and ‘Beaumont’ kernels roasted at 50 °C had a high concentration of antioxidants (0.017 and 0.017 ABTS mg GAE kg−1 and 0.005 and 0.007 DPPH mg GAE kg−1). Ref. [The decrease in flavonoids and phenols was accompanied by changes in antioxidant activity . ‘A4′ an1). Ref.  reported1). Ref. . It is c1). Ref.  showed t1). Ref. ,66. Thisp < 0.001) more crispy, which is thought to be a fundamental property of roasted kernels [As the roasting temperature increased, ‘A4′ and ‘Beaumont’ kernels became considerably  improved the color of the kernels  improved kernel flavor  had slightly elevated levels of flavonoids, phenols, and antioxidants, they also had higher moisture levels and FA composition, which promoted the development of rancidity, as indicated by higher levels of PV and poor kernel texture, color, and taste, as noted by trained panelists. The chemical and sensory qualities of macadamia nuts were greatly impacted by increasing roasting temperatures. ‘A4′ and ‘Beaumont’ kernels roasted at 125 °C had lower levels of rancidity (PV), high levels of oxidation-resistant FA compositions such as oleic acid, which delayed the rancidification process, and low levels of FA compositions, such as linoleic acid, which accelerated the oxidation process. Flavonoids, phenols, and antioxidants were significantly concentrated in the roasted kernels at 125 °C as well. Additionally, the judges said that roasted kernels at 125 °C had a perfectly crisp texture, a brown color, and were very nutty. However, a significant amount of rancidity; decreased content of flavonoids, phenols, and antioxidants; and an excessively crispy texture, a dark brown color, and a bitter flavor were present in kernels that were roasted at 150 °C. The results of this study, therefore, recommend roasting ‘A4′ and ‘Beaumont’ kernels at 125 °C to prevent rancidity and quality loss due to over-roasting. Additionally, because macadamia kernels quickly acquire rancidity, it is not advised to keep them fresh.


The phenyl ring is a basic structural element in chemistry. Here, we show the design, synthesis, and validation of its new saturated bioisostere with improved physicochemical properties − 2-oxabicyclo[2.2.2]octane. The design of the structure is based on the analysis of the advantages and disadvantages of the previously used bioisosteres: bicyclo[1.1.1]pentane, bicyclo[2.2.2]octane, and cubane. The key synthesis step is the iodocyclization of cyclohexane-containing alkenyl alcohols with molecular iodine in acetonitrile. 2-Oxabicyclo[2.2.2]octane core is incorporated into the structure of Imatinib and Vorinostat (SAHA) drugs instead of the phenyl ring. In Imatinib, such replacement leads to improvement of physicochemical properties: increased water solubility, enhanced metabolic stability, and reduced lipophilicity. In Vorinostat, such replacement results in a new bioactive analog of the drug. This study enhances the repertoire of available saturated bioisosteres of (hetero)aromatic rings for the use in drug discovery projects. The phenyl ring is a basic structural element in chemistry. Here, the authors show the design, synthesis, and validation of 2-oxabicyclo[2.2.2]octane as a new saturated bioisostere with improved physicochemical properties Moreover, more than five hundred drugs contain a fragment of para-substituted phenyl ring  is 1.8 Å, which is ca. 35% shorter than that in the para-substituted phenyl ring (2.8 Å). Bicyclo[2.2.2]octane has a closer C-C distance (2.6 Å), but higher lipophilicity41. Cubane, in turn, was recently demonstrated to be unstable under contact with transition metals43, under mechanochemical treatment or heating44.In 2012, however, Stepan and colleagues showed that a replacement of the central phenyl ring in a γ-secretase inhibitor with the bicyclo[1.1.1]pentane improved its physicochemical properties and retained bioactivityane Fig. 20–22. ThIn this work, we have rationally designed, synthesized, and characterized the new bioisostere of the phenyl ring – 2-oxabicyclo[2.2.2]octane Fig. .46 and in medicinal chemistry49 as an analog of 4-aminopiperidine53 or cyclohexane55. Also, 2-oxabicyclo[2.2.2]octane containing molecules exhibited a broad range of biological activities: estrogen receptor-beta agonists47, myeloperoxidase inhibitors48, antibacterial agents53, DGAT1 Inhibitors54, and RORγt agonists55.Interestingly, 2-oxabicyclo[2.2.2]octane core has been known in the literature, but not in the context of phenyl bioisostere. Chemists used it as a starting material in organic synthesis;φ = 180°). At the same time, we needed to address their drawbacks: C-C distance, and lipophilicity. Considering the possible saturated structures  in 15 steps using alkylation as a key reaction . However, under analogous conditions the expected product 6 was not formed , aromatic (13–17), and heterocyclic (18–27) aldehydes gave the corresponding 2-oxabicyclo[2.2.2]octanes in good yields. Functional groups such as nitro, trifluoromethoxy, trifluoromethyl, nitrile, and halogen atoms tolerated the reaction conditions. The protocol was not without limitations, however. We could not obtain products 28, and 29 with thiazole and triazole heterocycles, due to the formation of complex mixtures  and N-Boc acidic deprotection gave amino alcohol 59. Oxidation of the alcohol group in 58 gave N-Boc protected amino acid 60 – a saturated analog of the para-aminobenzoic acid. The reaction of iodide 57 with NaN3 (via azide 61) followed by reduction of the azide group formed diamine 62. The reaction of iodide 6 with NaN3 (via azide 63), the subsequent reduction (via 64), N-Boc protection, and saponification gave another N-Boc protected amino acid 65. The Curtius reaction of the latter provided N-Boc diamine 66 – isomer of diamine 62. The reaction of iodide 6 with sodium azide followed by extensive reduction of the intermediate azide with LiAlH4 gave amino alcohol 67. The structure of 67 was confirmed by X-ray crystallographic analysis (Supplementary Data 6 with potassium acetate (via 68) followed by saponification of the ester group and oxidation gave linker 69. Its structure was also confirmed by X-ray crystallographic analysis  in the presence of N,N’-diisopropylcarbodiimide (DIC) gave the activated ester 74 . The distance d between substituents in 2-oxabicyclo[2.2.2]octanes was also ca. 0.3 Å shorter than that in the para-phenyl ring: 5.56–5.58 Å vs 5.90–5.93 Å (para-phenyl). The difference in collinearity of vectors was insignificant, as angles φ1 and φ2 were almost identical in both scaffolds: 176-177° vs 178-179° (para-phenyl). Interestingly, even in the para-substituted phenyl ring in Imatinib in the crystal phase, the observed angles φ1 and φ2 deviated from the ideal value of 180°: 176-179°. It must be noted, that all parameters, - r, d, φ1 and φ2, - were also almost identical in both bicyclo[2.2.2]octanes (80–82) and 2-oxabicyclo[2.2.2]octanes   Fig. .para-substituted phenyl ring, as the geometric parameters r, d, φ1, and φ2 remained very similar  as a reference . However, analogous replacement at the β-position increased the acidity even more to pKa = 4.1 (54).We also studied the influence of the replacement of the methylene group for an oxygen atom in the bicyclo[2.2.2]octane skeleton on the electronic properties. Towards this goal, we measured experimental pKnce Fig. . Replace83 and 2-oxabicyclo[2.2.2]octane 47 were almost identical  vs 5.6 (84). However, incorporation of the β-oxygen atom into the latter ideally restored it: pKa = 4.4 (47). Because the acidity/basicity of functional groups is often responsible for the potency, selectivity, and toxicity of bioactive compounds65, the fine-tuning of the pKa by replacing the phenyl ring with isomeric 2-oxabicyclo[2.2.2]octanes could become a preferred solution.Important to mention that the acidity of aromatic carboxylic acid cal Fig. . The reppara- and mono-substituted phenyl rings, correspondingly  instead of the ly Figs.  and  8.F56 with N-methyl piperazine, followed by acylation with the substituted aniline gave compound 85 – a saturated analog of Imatinib . The commercialized drug Imatinib is used in practice as a mesylate salt. However, to estimate the impact of the replacement of the phenyl ring with bioisosteres on the physicochemical properties, we prepared and studied all three compounds, - 85,86, Imatinib, - as free bases.The reaction of iodide nib Fig. . For com87, in three steps we synthesized compound 88 - a saturated analog of Vorinostat .From amine para-substituted phenyl ring in Imatinib by bicyclo[2.2.2]octane (86) decreased the water solubility by more than three times  in Imatinib increased the solubility close to the original values: 351 µM (Imatinib) vs 113 µM (86) vs 389 µM (85).Replacement of the mes Fig. . However66 and experimental (logD) lipophilicities. Incorporation of bicyclo[2.2.2]octane in sted of the phenyl ring resulted in a decrease of clogP: 4.5 (Imatinib) vs 3.6 (86). The incorporation of 2-oxabicyclo[2.2.2]octane led to an even further decrease of clogP: 2.6 (85). A somewhat similar trend was observed with the experimental lipophilicity, logD. While the incorporation of the bicyclo[2.2.2]octane core into Imatinib did not significantly affect it; incorporation of the 2-oxabicyclo[2.2.2]octane core reduced it by ca. one unit, logD: 2.6 (Imatinib) vs 2.7 (86) vs 1.8 (85).To study the replacement of the phenyl ring with saturated bioisosteres on lipophilicity, we used two characteristics: calculated (clogP)86) and 2-oxabicyclo[2.2.2]octane (85) into Imatinib, increased the metabolic stability in human liver microsomes: CLint (mg/(min•μL)) = 28 (Imatinib) vs 16 (86) vs 19 (85)  into Imatinib increased the life half time by almost 50%: t1/2 (min) = 60 (Imatinib) vs 87 (85).The effect of saturated bioisosteres on metabolic stability was studied next. The incorporation of both bicyclo[2.2.2]octane (85) Fig. . Moreovepara-substituted phenyl ring in Imatinib with common bicyclo[2.2.2]octane core (86) led to an undesired three-times decrease in water solubility. At the same time, analogous replacement with 2-oxabicyclo[2.2.2]octane (85) resulted in an improvement of all measured physicochemical parameters: increased solubility, enhanced metabolic stability, and reduced lipophilicity.In summary, the replacement of the 85,86; and Vorinostat versus its analogs 88,89.Finally, to answer a key question, - whether the 2-oxabicyclo[2.2.2]octane core could indeed mimic the phenyl ring in bioactive compounds, we measured the biological activity of Imatinib versus its analogs 85,86 on the catalytic activity of ABL1 kinase. While the expected activity of Imatinib and Staurosporine was confirmed; we did not observe any significant inhibitory effect of compounds 85,86 on the ABL1 kinase .These primary biological results Fig.  suggestepara-substitued phenyl ring with 2-oxabicyclo[2.2.2]octane affects 3D-shape of organic compounds, we generated two virtual libraries based on C- and N-terminus modifications of para-aminobenzoic acid and its 2-oxabicyclo[2.2.2]octane-containing analog. Each library contained 5000 molecules : conformational rigidity, metabolic stability, non-chirality, and collinearity of the exit vectors Fig. . In addi.1.1pentaing Fig. . 2-Oxabily Figs.  and 8. Iity Fig. . In the rug Fig. .This study enhances the repertoire of available saturated bioisosteres of (hetero)aromatic rings for use in drug discovery projects.5  in MeCN (4000 mL) were added NaHCO3  in one portion and I2  in four portions. The resulting mixture was stirred for 12 h at room temperature. Then sodium thiosulfate pentahydrate  and distilled water (2000 mL) were added to the mixture. The colorless mixture was extracted with MeOtBu (10 × 400 mL). The combined organic layers were concentrated under reduced pressure to dryness. The residue was dissolved in MeOtBu (1000 mL), washed with brine (1 × 400 mL), a saturated solution of Na2S2O3 (3 × 400 mL), dried over Na2SO4, filtered through a plug of SiO2 (0.5 L glass filter filed with 3 cm in high with silica gel) and concentrated. The final product was purified by column chromatography  to provide pure iodide 6. Yield: 135.16 g, 0.436 mol, 36%, white solid.To a solution of alkene NMR spectra were analyzed with MestreNova (11.0.3-18688).Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Reporting SummarySource Data


Two bacterial strains able to use syringol as a sole carbon source were isolated from compost. The isolates, named S1 and S4, were sequenced using the Illumina platform. The final assemblies contained 4.2 Mbp, 63% GC, and 3,912 genes for S1 and 6.2 Mbp, 64% GC, and 5,503 genes for S4. Syringol is a major depolymerization product from lignins rich in sinapyl alcohol units (S-units), such as hardwood, cornstalk, and wheat straw lignin –3. Compa–Coarse-grained compost was provided by the Sysav waste company . Isolation was performed by adding 52.9 g compost to 200 mL NaCl , and vortexing vigorously. After settling, 1 mL supernatant was used to inoculate 250-mL shake-flasks with 25 mL M9 medium  supplemeGenomes from cells cultivated overnight  were extracted using the GeneJET genomic DNA purification kit . Sequencing was performed at Eurofins Genomics Europe (Germany) using Eurofins’ NGSelect DNA library preparation and the Illumina NovaSeq 6000 S2 PE150 XP platform. The total number of reads was 10,282,622 for strain S1 and 11,937,525 for S4. Read quality and trimming were performed with FastQC v0.11.9 . CoveragN50 of 428,008 bp, and 686× ± 115× average coverage. The S4 assembly had 6.18 Mbp, 33 contigs, a 64.19% GC content, an N50 of 370,688 bp, and 541× ± 90× average coverage. No plasmids were successfully assembled. Based on an average nucleotide identity analysis (Stutzerimonas sp. strain S1 and Achromobacter spanius S4. Totals of 3,912 and 5,503 protein-encoding genes were predicted in the S1 and S4 annotations, respectively. Putative genes from aromatic metabolic pathways were identified in both isolates, including beta-ketoadipate, ferulate, and syringate pathways. Syringol metabolism in bacteria is not well understood, but homologs to a recently described engineered guaiacol-demethylating cytochrome P450 system that enabled conversion of syringol to pyrogallol in Pseudomonas putida (The assembly of strain S1 resulted in 4.19 Mbp split over 22 contigs, a 63.08% GC content, an analysis , we propJAOEIY000000000 and JAOEJJ000000000, respectively. The versions described in this paper are JAOEIY010000000 and JAOEJJ010000000, respectively. The raw reads for S1 and S4 have been uploaded to the NCBI Sequence Read Archive as SRR21673156 and SRX17674200, respectively. The BioProject numbers for the data sets are PRJNA883164 and PRJNA883223.The annotated genome assemblies of strains S1 and S4 have been deposited at DDBJ/ENA/GenBank under the accession numbers


Background: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver diseases. In most cases, NAFLD progresses from benign steatosis to steatohepatitis (NASH), and then to cirrhosis. No treatment is currently approved for NAFLD/NASH in the clinic. Fenofibrate (FENO) has been clinically used to treat dyslipidemia for more than a half century, but its effects on NASH are not established. FENO’s half-life is quite different between rodent and human. The aim of this study was to investigate the potential of pharmacokinetic-based FENO regime for NASH treatment and the underlying mechanisms.Methods: Two typical mouse NASH models, methionine-choline deficient (MCD) diet-fed mice and choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice, were used. MCD model was designed as therapeutic evaluation in experiment 1 and CDAHFD model was designed as preventive in experiment 2. Three doses of FENO , two times a day (BID), were administered to the above models. Serum markers of liver injury, cholestasis, and the histology of liver tissues were investigated. Normal mice were used as a model in experiment 3 for toxicity evaluation, Quantitative-PCR and Western Blot assays were used to investigate the inflammatory responses, bile acid synthesis as well as lipid catabolism.Results: Mice on the MCD and CDAHFD diets developed steatohepatitis as expected. Treatment with FENO (25 mg/kg·BID) significantly decreased hepatic steatosis, inflammation and fibrosis in both therapeutic and preventive models. In the MCD model, the therapeutic action of FENO (25 mg/kg·BID) and 125 mg/kg·BID on histopathology and the expression of inflammatory cytokines were comparable. In reducing macrophage infiltration and bile acid load, FENO (25 mg/kg·BID) was superior to 125 mg/kg·BID. In all the aspects mentioned above, FENO (25 mg/kg·BID) was the best among the 3 doses in the CDAHFD model. In a third experiment, the effects of FENO (25 mg/kg·BID) and 125 mg/kg·BID on lipid catabolism were comparable, but 125 mg/kg·BID increased the expression of inflammatory factors and bile acid load. In both models, FENO (5 mg/kg·BID) showed little effect in hepatic steatosis and inflammation, neither the adverse effects. FENO (125 mg/kg·BID) aggravated liver inflammation, increased bile acid synthesis, and promoted the potential of liver proliferation. In toxicity risk assay, FENO (25 mg/kg·BID) treatment showed low potential to trigger bile acid synthesis, inflammation and hepatocyte proliferation.Conclusion: A new regime, FENO (25 mg/kg·BID) is potentially a therapeutic strategy for the NASH treatment. Translational medicine is warranted to prove its effectiveness in the clinic. Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic disease with complex etiological and pathophysiological backgrounds, characterized by prominent hepatocyte steatosis. The reported prevalence of NAFLD is 25%–30%, putting global public health at high risk . In ChinPeroxisome proliferator activated receptor alpha (PPARα) is a nuclear receptor that binds fatty acids and their derivatives. It regulates the expression of genes involved in fatty acid metabolism in the liver . ClinicaSome groups reported their efforts trying FENO therapy for NAFLD. Studies showed FENO improved liver function, liver steatosis, and inflammatory response in NASH patients and mice. In high-fat diet (HFD)-fed mice, FENO (100 mg/kg day) decreased serum alanine aminotransferase/aspartate aminotransferase (ALT/AST) levels and the amount of liver fat . In methFENO therapy, however, was also found to worsen hepatic steatosis and increased the inflammatory responses in NASH in several studies. FENO (100 mg/kg·day) demonstrated only minimal improvements in hepatic steatosis in NASH mice . At a daControversial conclusion about FENO therapy for NASH might be attributed to protocol design. In this study, three doses of FENO  were administered intragastrically, two times a day , to evaluate their actions on methionine-choline deficient (MCD) and choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-induced NASH models. The MCD model was therapeutic in experiment 1, and the CDAHFD model was preventive treatment in experiment 2. Meanwhile, the toxicity risk of FENO treatment were evaluated in experiment 3. Conclusion were made based on combination of pathological phenotype, anti-inflammation, biochemistry and toxicity risk.FENO was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Sodium carboxymethyl cellulose (CMC-Ma) was obtained from Sangon Biological Co., Ltd. The detection kits for total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) were acquired from Ningbo Meikang Biotechnology Co., Ltd. Trizol was purchased from Ambion Invitrogen in the United States. Hematoxylin-eosin (HE) and Masson-Trichrome (MT) staining kits were purchased from Shanghai Sobo Biotechnology Co., Ltd. Reverse transcription and quantitative PCR (Q-PCR) kits were produced by Beijing Kangwei Century Biotechnology Co., Ltd. F4/80 antibody was purchased from Abcam . BCA Protein Assay Kit was purchased from Beyotime Biotechnology Co., Ltd. Antibodies against total protein 38 (t-p38), phosphorylated protein-38 (p-p38), total extracellular regulated protein kinases 1/2 (t-ERK1/2) and phospho-extracellular regulated protein kinases 1/2 (p-ERK1/2) were obtained from ABclonal Technology Co., Ltd. Total JNK (t-JNK) and phospho-JNK (p-JNK) were bought from Cell Signaling Technology Co. Ltd. Total extracellular regulated protein kinases 5 (t-ERK5) and phospho-extracellular regulated protein kinases 5 (p-ERK5) were purchased from Santa Cruz Biotechnology Co., Ltd. The goat anti-mouse IgG and goat anti-rabbit IgG were bought from Proteintech Co., Ltd.MCD diet  contains : amino acid premix (methionine free) 175.7 g, sucrose 431.9 g, dextrin 50 g, corn starch 150.0 g, corn oil 100.0 g, cellulose 30.0 g, mineral mix 52.4 g. CDAHFD diet  contains  amino acid premix 349.2 g, methionine 1.6 g, sucrose 345.6 g, corn starch 154.2 g, soybean oil 50.0 g, cellulose 100.0 g, mineral mix 20.0 g, vitamin mix 20.0 g.ad libitum for 1 week. After 1 week of acclimatization, the mice were randomly assigned into three experiment plans.Seventy male C57BL/6 mice (SCXK (Shanghai) 2017-0005) were obtained from Shanghai Slack Laboratory Animal Co., Ltd., which were 7 weeks old and weighed 23–25 g. Mice were allowed access to normal chow and water In the first experiment, the mice were divided into five groups: MCD-CON group; MCD group; MCD-FENO  groups (n = 5). Mice were fed the MCD for 5 weeks to establish a NASH model . After 5In the second experiment, the mice were divided into five groups: CDAHFD-CON group; CDAHFD group; CDAHFD-FENO  groups (n = 5). CDAHFD diet was used to treat the diet and FENO  was orally gavaged concurrently for preventative therapy for 3 consecutive weeks. The body weight of the mice was measured every week during the experiment.In the third experiment, 20 mice were divided into four groups: CON group; F5, F25, F125 groups. The CON group was treated with normal diet. F5, 25, 125 groups were treated with 5, 25, 125 mg/kg FENO, twice a day by oral gavage for 5 consecutive days. The aim of experiment 3 was to evaluate the toxicity risk of FENO. The expression of inflammatory factors, lipolysis factors and bile acid loading proteins were examined. FENO was administered two time everyday, at 8 a.m. and 8 p.m., with a 12-h interval between doses in three experiments. FENO was dissolved in 1% CMC-Na for administration.All CON groups in 3 experiments  were treated with 1% CMC-Na by oral gavage.The body weight of mice was measured weekly. Body weight and liver wet weight were measured when mice were sacrificed. The ratio of liver wet weight to body weight (LW/BW) was calculated as the liver index. Blood samples were collected after the mice were sacrificed. They were centrifuged at 3,600 rpm for 15 min at 4°C to get the plasma. The supernatant was collected and stored at −80°C. Half of the biggest liver lobe was excised, fixed in 10% formalin, dried, and embedded in paraffin. The leftover of liver tissues was flesh frozen and kept at −80°C pending analysis.Biomarkers TC, TG, ALT, AST, TBA and ALP in serum were assayed using the Multiskan GO . The analyses were carried out following the manufacturers’ procedures described in the kits.20 mg of liver tissues were mixed with 180 μL of saline in a tissue homogenizer for 30 s at 4°C. Liver tissues were treated at 4°C at 13,000 rpm for 20 min, then the supernatant was transferred to an Eppendorf tube devoid of enzymes. Liver TC and TG were assayed using the Multiskan GO . The analyses were carried out following the same protocols as the above serum biomarkers.Fixed liver tissues in the above experiments were dehydrated in a serial concentration of alcohol and xylene, followed by paraffin embedding. Four-micrometer serial sections were cut and stained with hematoxylin-eosin (HE) and Masson-Trichrome (MT). Histopathological examination was performed using an Olympus BX51light microscope . The non-alcoholic fatty liver activity score (NAS) was assessed based on HE staining . The sco2O2 for 5–10 min at room temperature before being washed with distilled water. After 5 min in phosphate buffer saline (PBS), the sections were blocked in 5%–10% PBS-diluted normal goat serum. Finally, the sections were incubated at 22°C for 10 min. Then, the primary antibody (F4/80) was added for 15 min of incubation at room temperature. A secondary antibody was added for 10–30 min incubation at 37°C and rinsed with PBS for 5 min × 3 times. Chromogenic agent 1, 2-diacetylbenzene (DAB) was used for development and visualization. Photomicrographs of liver tissue sections were taken using an Olympus BX51light microscope and positively immunostained area was quantified with Image J software.Paraffin-embedded liver sections (4 μm thickness) were deparaffinized in xylene and rehydrated in a gradient of ethanol to distilled water. Sections were incubated in 3% Hhttps://pga.mgh.harvard.edu/primerbank. Each 10 μL PCR system contained 1 μL total cDNA, Light Cycle 480 SYBR Green I Master Mix  5 μL, forward primer 0.2 μL, reverse primer 0.2 μL, nuclease-free water 3.6 μL. Amplification was performed using a reaction cycle at 95°C 10°s, 55°C 10 s and then 72°C 15 s. The fluorescence signal was detected at the end of each cycle. 18S rRNA was used as an internal control, and 2-△△Ct was used to calculate the expression of the target genes. Primers for Ccl2, Cxcl2, Tnf-α, Il-10, Cyp4α10, Acot1 and Cpt1 were shown in The total RNA from mice hepatic tissues (20 mg) homogenized in Trizol reagent were determined by Multiskan Go . The reverse transcription system (20 μL) included the following items: 5× Reaction buffer 4 μL, total RNA 1 μg, Oligo dT18 1 μL, Random primer 1 μL, 10 mM dNTPs Mix 2 μL, 200 U RevertAid M-MuLV RT 1 μL and 20 U Ribolock RNase inhibitor 1 μL. The cDNA synthesized was stored at 20°C and subjected to analysis within 7 days. The primer sequences were extracted from The fresh liver tissue was lysed by RIPA and PMSF at a ratio of 100:1. The insoluble material was removed by centrifugation  for 20 min at 4°C. The protein concentration in the supernatant was determined by the BCA protein assay kit and then adjusted to 8 mg/mL. Then, 5 μL of each sample was loaded using SDS-polyacrylamide gels and transferred to PVDF membranes after electrophoresis. PVDF membranes were blocked with 5% skimmed milk for 3.5 h at room temperature. Next, membranes were incubated with primary antibodies overnight at 4°C. Finally, the blotted PVDF membranes were incubated with secondary antibodies for 2 h at room temperature after being washed 3 times using TBST. The washed membranes were exposed to ECL substrates  and the signals were detected by Tanon 4200SF . The bands were quantified using Image J software.one-way ANOVA followed by Dunnett’s post hoc comparisons. Comparison between normal and model groups was tested by Student’s t-test. The difference was considered significant when p < 0.05 and was marked with an asterisk in the graphs accordingly.The assignment of mice to different groups was randomized. All the data were presented as mean ± SD. The statistical analysis was performed using the software SPSS Statistics, version 25 . Differences among multiple groups were tested using p< 0.001). The body weight decreased in MCD-FENO groups and there was no significant difference among the 3 MCD-FENO groups . In group MF-25, the above indicators were inhibited by 63% and 25% respectively (p < 0.001 and p = 0.044). In the MF-5 and MF-125 groups, serum ALT was reduced by 60% and 73% respectively  in the CDAHFD group. In the group CF-25, compared to theh CDAHFD group, serum ALT and AST concentrations were decreased by 54% and 55% (p < 0.001). Compared to the CDAHFD group, serum ALT in the CF-5 group and the CF-125 group was increased by 22% and 26%, respectively. In the CF-5 and CF-125 groups, serum AST was only decreased by 3% and 14%  and 39% (p = 0.001) in the MF-5, 25, 125 groups, respectively. Compared with CDAHFD group, the NAS score decreased by 3%, 56% (p < 0.001) and 19% (p = 0.003) in the CF-5, 25, 125 groups, respectively. In two NASH models, the decrease in NAS scores was statistically significant at FENO 25 and 125 mg/kg·BID  treatment, the lipid accumulation of the liver was significantly reduced in two models . Compareg/kg·BID .MT staining revealed significant fibrotic changes in the liver tissue of the MCD model. Hepatic collagen deposition was seen in the MCD and MF-5 groups, indicating the formation of liver fibrosis. Hepatic collagen deposition was reduced in MF-25 and MF-125 groups . There wp = 0.035), suggesting lipid deposition in the liver. After FENO treatment, the liver TG levels of the MF-5, MF-25, and MF-125 groups decreased by 27%, 50% (p = 0.024), and 60% (0.007), respectively. For the liver TC, there was no significant difference amount the 5 groups (p < 0.001). But the liver TC was reduced by 82% in the CF-25 group (p = 0.019), which was the strongest among the 3 dose levels. Similarly, there were no significant changes in liver TC levels between the CDAHFD, CF-5, CF-25, and CF-125 groups , when compared to the MCD-CON group. The levels of Ccl2 mRNA in MF-25 and MF-125 groups decreased by 76.5% and 83.5%, respectively (p = 0.015 and p = 0.009). Cxcl2 level was decreased by 14%, 25%, and 51% (p = 0.035), respectively, in the MF5, MF25, and MF125 groups  in the MF-5, MF-25, and MF-125 groups, respectively. Il-10 level was reduced by 36%, 62% (p = 0.016), and 9% when compared to the MCD group after FENO treatment . In the CF-25 group, the above mRNA levels were significantly reduced after being treated with the regime of FENO (25 mg/kg·BID), which was also the strongest among the 3 dose levels. The difference was statistically significant in comparison to the CDAHFD group , suggesting that the mice developed hepatic cholestasis. Serum ALP in the MF-5 group treated with FENO showed slight improvement. In the MF-125 group, serum TBA and ALP were 2-fold higher than those in the MCD group . In the CF-25 group, serum TBA levels dropped by 46% (p < 0.001). Importantly, serum ALP decreased by 20% and 15% in the CF-5 and CF-25 groups, but increased by 30% in the CF-125 group . Compared to the MCD group, the liver index of the MF-5 and MF-25 groups did not change significantly, but the liver index of the MF-125 group doubled . CF-5, CF-25, and CF-125 dose groups all increased the liver index. Following FENO treatment, the liver index was decreased by 4% and 24% (p < 0.001) in the CF-5 and CF-25 groups, respectively, but was elevated 13% in the CF-125 group compared to the CDAHFD group  improved insulin resistance, cirrhosis, and fibrosis in NAFLD . Animal The above low dose dependence of FENO therapy was remarkable and deserved investigation. Pharmacokinetically, FENO had a half-life of approximately 20 h in humans and 6.8 h in mice . BesidesIn our study, as a therapeutic model, the MCD model showed a significant body weight reduction. The CDAHFD model, as a preventive treatment model, showed a steady body weight change. There was no appreciable difference in body weight in two models following 3 doses of FENO treatment. And there was a significant decrease in serum ALT, AST and inflammatory factors levels in term of liver injury and inflammation. Histopathologically, the MCD and CDAHFD models exhibited steatosis and inflammatory cell infiltration, with the greatest improvement in NASH occurring at 25 mg/kg·BID. However, the CDAHFD model showed no fibrotic modifications, which may be due to the short time of modeling and no fibrosis development. In both therapeutic and preventive treatment models, FENO (125 mg/kg·BID) led to higher serum TBA and ALP levels and liver inflammatory markers than FENO (25 mg/kg·BID). Experiment 3 therefore demonstrated the toxicity risk of FENO alone on bile acid metabolism, lipid metabolism, and inflammation markers.Cyp4a-10, Acot1, Cpt1 and Ehhadh) were activated after FENO treatment between MCD and CDAHFD models (data are not shown). However, there was no significant difference between 25 and 125 mg/kg. In terms of liver inflammation, ALT and AST are primarily produced by liver and cardiac tissues, particularly liver cells. When the liver cells and mitochondria were be injured, they would be released into the bloodstream, resulting in a significant rise in serum ALT and AST levels. The inflammatory factors Ccl2, Cxcl2, Tnf-α and Il-10, as upstream of inflammation, are more sensitive than ALT and AST. Ccl2 and Cxcl2 are chemokines that function in recruiting neutrophils. When tissues were challenged, chemokines mediated the occurrence and development of inflammatory responses  reduced lipid accumulation and also delivered anti-inflammatory benefits.PPARα is a nuclear receptor that, upon activation, increases expression of a multitude of genes involved in fatty acid oxidation . In our esponses . Tnf-α ith NAFLD . It can th NAFLD . Il-10 wnd Tnf-α . In thisCcl2, Tnf-α, Il-10, Cyp4α10, Acot1 and Cpt1) increased in a dose-dependent manner. These factors were used as sensitive indicators rather than phenotypic ones. The lipolytic effects of FENO (25 mg/kg) was comparable to that of 125 mg/kg. FENO at 125 mg/kg, however, was found to increase the bile acid synthesis and inflammatory response. Above all, although the adverse effects of FENO (5 mg/kg·BID) in this trial were minor, the therapeutic benefits were negligible. FENO (125 mg/kg·BID), on the other hand, triggered inflammatory response, an increase of bile acid load, as well as a proliferative response in the liver. Thus, FENO (25 mg/kg·BID) was the most efficient in alleviating NASH.FENO also caused many risks in the clinic. In hyperlipidemic patients, FENO increased ALT and AST levels relating to liver function . Long-teThere were several limitations in our study. First of all, CDAHFD is a model with severe steatosis and inflammation, which exceeded the reversible therapeutic check point quickly. Thus, in our study, CDAHFD was only used as a preventative model. Secondly, translational medicine remained to be investigated following these animal experiments. Finally, the MAPK family includes four primary pathways. It remains to be investigated to explore which pathway mediated the anti-inflammatory effect and what the underlying molecular mechanisms were.Conclusively in this study, FENO (25 mg/kg·BID) treatment mitigated NASH by inhibiting hepatic steatosis, inflammation and fibrosis in both therapeutic and preventive treatment models, which stemmed from its action on lipid catabolism and anti-inflammation. FENO (5 mg/kg·BID) showed little effect in hepatic steatosis and inflammation, neither the adverse effects. FENO (125 mg/kg·BID) aggravated liver inflammation, increased bile acid load, and promoted the potential of liver proliferation. Also, the toxic risk of high-dose FENO were confirmed in the normal mice model. Considering species differences in pharmacokinetics and the low risk of dose-dependent adverse reactions, translational medicine is warranted to prove the effectiveness of FENO (25 mg/kg·BID) in the clinic.


Replication factor C subunit 2 (RFC2) participates in the growth and metastasis of various malignancies. Our study investigated the roles of RFC2 in colorectal cancer (CRC).RFC2 expression was upregulated in CRC tissues and cells. High RFC2 expression was associated with poor prognosis. Knockdown RFC2 inhibited proliferation, induced apoptosis, and suppressed migration and invasion of CRC cells. CREB5 was a transcription factor of RFC2, and CREB5 knockdown suppressed RFC2 expression. Furthermore, RFC2 promoted aerobic glycolysis and MET/PI3K/AKT/mTOR pathway.RFC2 promoted the progression of CRC cells via activating aerobic glycolysis and the MET/PI3K/AKT/mTOR pathway.The online version contains supplementary material available at 10.1186/s12876-023-02984-0. Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide . China aReplicate factor C (RFC) is a primer recognition factor of DNA polymerase, which is reported to participate in DNA replication and repair . The RFCCREB5, a transcription factor (TF), interacts with the promoter of genes, and promotes the progression of cancers , 23. OurIn this study, we explored the expression and prognostic value of RFC2 in CRC. We sought to investigate the underlying mechanisms of RFC2 in CRC.http://gepia2.cancer-pku.cn/#index). The survival analysis of RFC2 expression in patients with CRC was analyzed using Kmplot . RFC2 expression was performed gene set enrichment analysis (GSEA) using R package.Two hundred seventy-five colon adenocarcinoma and 349 normal tissues were collected from TCGA-COAD dataset .Normal human colonic epithelial cells (FHC) and five human CRC cell lines  were purchased from Nanjing Cobioer Biotechnology Co., LTD. FHC cells were cultured in DMEM/F12  containing 10% fetal bovine serum (FBS), cholera toxin (10 ng/ml), insulin (0.005 mg/ml), transferrin (0.005 mg/ml), hydrocortisone (100 ng/ml), and human recombinant EGF (20 ng/ml). HCT116 cells were cultured in McCoy's 5a containing 10% FBS. DLD-1 cells were cultured in 1640 containing 10% FBS. LoVo and SW480 cells were cultured in DMEM containing 10% FBS. Caco-2 cells were cultured in MEM containing 20% FBS. All cell lines were maintained in a humidified chamber containing 5% CO2 at 37 °C.The overexpression plasmid of RFC2 and CREB5 (oe-RFC2 and oe-CREB5), small interfering RNA of RFC2 and CREB5 (si-RFC2 and si-CREB5), and their negative controls were obtained from GenePharma . Lipofectamine™ 3000 (Invitrogen) was used for above plasmids transfection.−ΔΔCt method. RFC2, forward primer: CTACGAACTGCCGTGGGTT, reverse primer: GAGGGCCCGCAATGATGATG. β-actin, forward primer: AACACCCCAGCCATGTACGTT, reverse primer: CCATCTCTTGCTCGAAGTCCA.The TRIzol RNA reagent  was used to extract RNA from cells. The HiScript II Q Select RT SuperMix  was employed to make cDNA. SYBR green  was employed to real-time PCR analysis. RT-qPCR was performed using an ABI StepOnePlus real-time PCR system (Applied Biosystems). The expression of RFC2 was determined by 24 cells/well) were seeded in 96-well plates. At 0, 48, and 72 h, 10 µl of CCK-8 reagent  was added to each well for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader .Cells (2 × 10Cells were suspended in buffer and stained with FITC Annexin V Apoptosis Detection Kit (Beyotime) in dark place for 10 min. Apoptosis was measured using flow cytometry (BD Biosciences).For wound healing assays, until 95% of the cells covered the 6-well plate, wound was produced by scratching the cell monolayer with 10 μl pipette tips. Wound images were taken with a microscope at 0 and 24 h. For invasion assays, Matrigel (BD Biosciences) was added to the upper chamber. Cells were shifted to the upper chamber, while medium containing 10% FBS was added to the lower chamber. After 24 h, cells were stained with crystal violet solution for 2 h. The pictures were taken under a microscope .The assay was performed using ChIP Assay Kit . Chromatin was immunoprecipitated using polymerase II, CREB5, or IgG antibody. RFC2 expression was evaluated using RT-qPCR.Wild type (wt) and mutant type (mut) promoter region of RFC2 were cloned into pGL3-report vector (GenePharma). HEK-293 T cells were co-transfected with RFC2-wt, RFC2-mut, ov-CREB5, or ov-NC plasmids using Lipofectamine™ 3000 (Invitrogen). To examine luciferase activity, dual-luciferase reporter assay system (Promega) was used at 24 h post-transfection.Cells were lysed in RIPA, and protein concentration was detected by a BCA Kit (Beyotime). Proteins were separated by SDS-PAGE and further transferred onto PVDF membranes . Membranes were incubated overnight at 4 °C with RFC2 , CRBB5 , LDHA , GLUT1 , HK2 , p-PI3K , PI3K , p-AKTSer473 , 1:1000, 4060), AKT , p-mTOR , mTOR , p-70S6K , 70S6K , MET , and β-actin antibody . Membranes were incubated with secondary antibody . The proteins were detected using a GEL imaging system .Glucose uptake and lactate production were determined in the supernatants from the cells according to the protocols of Glucose Uptake Assay Kit and Lactate Assay Kit .p < 0.05 was defined as statistically significant.GraphPad Prism 8.0 software was used to conduct statistical analyses. The differences among groups were analyzed using the student’s t-test or one-way ANOVA. p = 0.033) was found to related with a poor prognosis of CRC (Fig. RFC2 expression was analyzed using TCGA dataset. RFC2 was upregulated in CRC tissues compared with normal tissues Fig. A. HigherCRC Fig. B. RFC2 pCRC Fig. C and D. CRC Fig. E. KaplanCRC Fig. F. ComparCRC Fig. G. RFC2 esi-RFC2 and oe-RFC2 were transfected in HCT116 and SW480 cells. The efficacy was validated using RT-qPCR and western blot (Fig. A sketch map of RFC2 promoter region was shown in Fig. By using GSEA analyzers based on RFC2 expression in CRC from the TCGA database, RFC2-enriched pathways are shown in Fig. Based on the GESA forecast, RFC2 activated the PI3K/AKT/mTOR pathway in CRC Fig. A. RFC2 e740Y-P attenuated the decreased cell viability, migration, and invasion caused by knockdown of RFC2. Furthermore, LY294002 led to a decrease in cell viability, migration, and invasion in RFC2-overexpression cells compared with untreated cells Fig. A-C. 740YRFC2 is famous for DNA replication and damage repair, and participates in disorder of biological process, such as Williams-Beuren syndrome, type 2 diabetes mellitus, and cancers –27. PrevTF is important regulators of intrinsic life processes within cells, possess DNA-binding domains and initiate transcription of genes . Over thAerobic glycolysis is a notable feature of malignant cancers, that enhances unrestricted tumor growth and metastasis . NormallMET regulates the proliferation, migration, and invasion of cancer cells by mediating the pathways such as PI3K/AKT pathway, MAPK pathway, and FAK pathway , 43. METIn order to better understand the mechanisms of RFC2 in CRC, pathways were enriched using GSEA. RFC2 expression is connected with the PI3K/AKT/mTOR pathway. Down-regulated phosphorylation of PI3K, AKT, mTOR, and 70S6K in CRC cells leads to inhibition of proliferation and aerobic glycolysis , 47. Li RFC2 expression was upregulated in CRC. RFC2 promoted proliferation, migration, invasion by affecting aerobic glycolysis and the MET/PI3K/AKT/mTOR pathway in CRC cells. Our research demonstrated that RFC2 might be a candidate gene for the therapy of CRC.Additional file 1.


Controlling the number of molecular switches and their relative positioning within porous materials is critical to their functionality and properties. The proximity of many molecular switches to one another can hinder or completely suppress their response. Herein, a synthetic strategy involving mixed linkers is used to control the distribution of spiropyran-functionalized linkers in a covalent organic framework (COF). The COF contains a spiropyran in each pore which exhibits excellent reversible photoswitching behavior to its merocyanine form in the solid state in response to UV/Vis light. The spiro-COF possesses an urchin-shaped morphology and exhibits a morphological transition to 2D nanosheets and vesicles in solution upon UV light irradiation. The merocyanine-equipped COFs are extremely stable and possess a more ordered structure with enhanced photoluminescence. This approach to modulating structural isomerization in the solid state is used to develop inkless printing media, while the photomediated polarity change is used for water harvesting applications. Controlling the number of molecular switches and their relative positioning within porous materials is critical to their functionality and properties. Here the authors systematically control the number of spiropyran units in a covalent organic framework using a mixed-linker synthetic strategy which resulted in one photoresponsive unit per pore. However, supramolecular self-assemblies are formed by weak non-covalent interactions that are easily destroyed by heat or changes in pH, effectively limiting their practical utility14. To overcome these problems, scientists have introduced the bottom-up self-assembly process for the synthesis of 2D nanosheets15, hollow spheres16, and tubular morphologies17 in covalent organic frameworks (COFs)18. These materials have attracted much attention due to their excellent chemical stability and strong mechanical properties15. Unfortunately, the preparation of COFs by the bottom-up self-assembly approach suffers from long reaction times and requires much effort to obtain product17.The self-assembly of organic nanostructured materials with tunable morphology has become a fascinating topic in material science and supramolecular chemistry22. This strategy is promising for those seeking to develop new well-defined nanoscale architectures in short time that also have potential applications in optics26, sensing29, and delivery systems32. Stimuli-responsive self-assembly of one- and two-dimensional supramolecular materials, including polyrotaxanes40, nanotubes42, nanowires48, and nanosheets52, has already been extensively studied53. However, the modulation of morphology in response to light has rarely been reported despite being one of the most environmentally friendly sources of external stimuli that is commonly accessible in our daily life55. Compared to other stimuli, light has numerous advantages because it can be applied at a specific location and in the form of different wavelengths57. An attractive strategy to control morphological self-assembly in response to light is to incorporate photoactive molecular switches that undergo structural isomerization upon light irradiation into the COF network60. Among the numerous photoactive molecular switches described in the literature, spiropyran (SP)61 is one of the best studied molecular switches that can reversibly photoisomerize between the hydrophobic ring-closed SP and the hydrophilic ring-opened merocyanine (MC) upon irradiation with visible or UV light63. Due to their excellent reversible reactivity, materials functionalized with spiropyran have been widely used for data storage65, chemical sensors69 and developing smart materials71. However, on account of the space confinement effect and/or their structural rigidity, it remains a challenge to achieve reversible photoisomerization in ordered structures such as metal-organic frameworks (MOFs)78 and COFs81. Recently, we developed a photosensitive quasi-honeycomb COF by introducing dangling azobenzene units into the pore structure58. The material exhibited interesting reversible photoswitching behavior due to the free movement of the azobenzene groups within the pores upon irradiation. Inspired by our previous work, we first attempted to intrinsically introduce spiropyran into a COF structure as a pendant group by following our reported synthetic protocol with the same 1:1.5 monomer:linker ratio. Interestingly, unlike the azobenzene COF, the spiropyran COF did not exhibit any photoswitching properties, which could be explained by the larger size of the spiropyran moiety as compared to the azobenzene unit, which hinders mechanical motion within the pores. To avoid the crowding effect, we used a mixed linker synthesis method  was prepared via imine condensation of the monomers 4,4′,4″--trianiline (TTA), pyridine-2,6-dicarbaldehyde (DFP), and (R)-4--pyridine-2,6-dicarbaldehyde  and the C=O stretching vibrations (1700 cm–1 for SPDFP and 1702 cm–1 for DFP) disappeared while a new characteristic C=N stretching band at 1500 cm–1 was detected, suggesting the formation of an imine bond. The 13C NMR spectra  of the acid-digested COF showed two distinct singlet signals at δ = 8.41 and 8.19 ppm with an integration ratio of 2:1, corresponding to the DFP and SPDFP subunits, respectively. The TTA protons (e and d) of the acid-digested TTA-SPDFP COF resonance signals appeared as two separate doublet signals at δ = 8.61 and 7.54 ppm, and the integration region of the TTA resonances showed a 2:2:1 ratio of TTA, DFP, and SPDFP compared with the precursor linkers. The acid-digested TTA-SPDFP COF also showed three characteristic aldehyde proton signals (c and f) at δ = 10.01, 10.10, and 9.95 ppm, and all protons had the same integration area (2:0.5:0.5), which corresponded to the aldehyde protons of DFP and SPDFP. Due to the ring opening of the SPDFP subunit in the strongly acidic medium, the symmetry of SPDFP was lost, so that the two aldehyde protons appeared as two separate singlets at δ = 10.10 and 9.95 ppm. Area integration of the remaining other protons (h-r) of the acid-digested TTA-SPDFP COF, corresponded to SPDFP, and showed good agreement with proton (a) of the DFP subunit. Together, these results support the formation of TTA-SPDFP with a linker ratio of 2:2:1 for the TTA, DFP and SPDFP subcomponents. Thermal stability of the as-synthesized COF was confirmed by thermogravimetric analysis (TGA), which revealed that the TTA-SPDFP is thermally stable up to 400 ± 5 °C  optimized unit cell parameters were found to be a = 33.0 Å, b = 33.0 Å, and c = 10.4 Å. The proposed crystal structure of TTA-SPDFP  analyses were performed on the TTA-SPDFP COF Fig.  and its 91 with an average diameter of 170 nm  spectroscopy. As shown in the inset of Fig. ers Fig. . Surpristra Fig.  of the T96, hence the two forms of spiropyran always coexist. This is a very well-known phenomenon in materials associated with spiropyran98. The spiropyran moiety is very sensitive to moisture, and some of the spiro moieties can be converted to the merocyanine form in the presence of moisture in the absence of UV light. The two-dimensional 1H-13C heteronuclear correlation spectra  was characterized by the presence of a 1H-13C correlation signal between 8 ppm (1H) and 193 ppm (13C), which is characteristic of the N⊕=C bonded C, as previously reported from similar solution-state NMR studies95. This signal was absent in the spectrum obtained for the closed state.The presence of a signal with a chemical shift of 193 ppm in the closed state suggested that some of the spiro moieties may have opened up when exposed to humiditytra Fig.  of TTA-STTA-SPDFP COF exhibits reversible photoinduced surface wettability Fig. . Water c2 adsorption isotherms at 77 K, as shown in Supplementary Fig. 2g–1 compared to the closed form, TTA-SPDFP, with a surface area of 334 m2g–1. As additional evidence of the conversion of SPDFP to MCDFP, non-local density functional theory calculations (NLDFT) show a reduction in the total pore volume from 0.28 cm3g–1 to 0.13 cm3g–1, respectively, though no drastic changes were observed in the average pore sizes, as shown in Supplementary Fig. The permanent porosity of TTA-SPDFP and TTA-MCDFP was determined by measuring N102. AFM analysis was also performed on dried samples in an effort to understand the vesicle formation  switched from non-polar to polar . This can be attributed to the higher degree of conjugation and the planar structure of the merocyanine isomer in the TTA-MCDFP COF. To obtain further details on the correlation between fluorescence enhancement and structural changes following light irradiation, HRTEM was recorded at a very high magnification to detect any profound structural transformation processes in response to UV light  and the pyridine-2,6-dicarbaldehyde (DFP)109 were synthesized following previously reported synthetic procedures. Thin-layer chromatography (TLC) was performed on 60 F254 silica gel TLC plates. The plates were inspected with UV light of wavelengths 256 nm and 302 nm. Column chromatography was performed using the Combi flash EZ Prep system. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III spectrometer, with working frequencies of 500 MHz for 1H, and 125 MHz for 13C nuclei at room temperature. All chemical shifts are given in ppm relative to the signals corresponding to the residual non-deuterated solvents (CHCl3 = 7.26 ppm)111. All 13C spectra were recorded with the real-time decoupling of proton nuclei. The values of the coupling constants (J) are given in hertz (Hz). The multiplicity of the proton spectrum is abbreviated as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), and m (multiplet). High resolution mass spectrometry (HRMS) studies were done using an Agilent 6540 UHA Accurate Mass Q-TOF/LC-MS-spectrometer in the ESI positive ion mode using an acetonitrile/water a gradient with C18 column. All solid-state NMR experiments were carried out on a Bruker Avance-HD 600 MHz spectrometer operating at a static field of 14.1 T, resonating at 150.0 MHz for 13C, using a 3.2 mm triple resonance MAS probe. Fourier transform infrared (FT-IR) studies were carried out on the Agilent 670-IR spectrometer. Thermogravimetric analysis (TGA) was performed on TA SDT Q600. Scanning electron microscopy (SEM) images were obtained from FEI Quanta 450FEG. The topography of the TTA-SPDFP/MCDFP COFs were analyzed by dynamic atomic force microscopy . We acquired topography, phase, and amplitude scans simultaneously. Silicon cantilevers  with resonant frequencies of 250–300 kHz and force constants of 100–130 Nm─1 were used. The set point value was maintained at 2.5 V. AFM scans were collected at 1024 points/lines with scan speed of 0.20 at fixed scan angle of 0o. Scan artifacts were minimized by acquiring a typical scan at an angle of 90o under identical image acquisition parameters. We used GwyddionTM free software (version 2.47), SPM data visualization and analysis tool for post-processing the AFM scans. Size and morphology of the COF material was determined with a TEM (FEI-Titan 300) microscope. Samples were prepared on a carbon-coated copper grid. A drop of dispersed TTA-SPDFP/MCDFP COFs network was spotted on the grid and allowed to dry overnight. Surface area measurements were conducted on a Micromeritics 3Flex gas sorption analyzer. Samples (20–40 mg) were degassed at 85 °C for 24 h and then backfilled with N2. Adsorption isotherms were generated by incremental exposure to ultrahigh-purity nitrogen up to 1 atm in a liquid nitrogen bath, and surface area was determined using BET adsorption models included in the instrument software (Micromeritics ASAP 2020 V4.00). Powder X-ray diffraction (PXRD) measurements were carried out using the PANalytical X’Pert PRO MP X-ray diffractometer consisting of a focusing elliptical mirror and a fast-high resolution detector (PIXCEL) with a radiation wavelength of 0.15418 nm. Solid state UV-vis studies were carried out on the Cary 5000 UV-vis-NIR spectrophotometer. Emission spectra in water at room temperature were recorded on a Perkin Elmer LS55 Fluorescence Spectrometer.All chemicals and reagents were purchased from Sigma-Aldrich and used without further purification. The 4-bromopyridine-2,6-dicarbaldehyde2 line; thus, N2 acts as a carrier gas for water vapor. Pre-drying of the sample was carried at 100 °C in the presence of N2 dry carrier. The sample “dry mass” was measured under N2 and was at equilibrium (25 °C) before dosing water vapor progressively into the chamber. The adsorption isotherms, obtained at equilibrium, were collected between 0 and 98 % RH at room temperature. The maximum equilibration time for each RH was maintained for 2 h.Water vapor adsorption experiments were carried out to examine water vapor adsorption characteristics of TTA-SPDFP /MCDFP COF using IGAsorp vapor sorption analyzer. The water vapor partial pressure was controlled automatically by mixing wet vapor feed with a dry N112 and optimized using Density Functional Tight Binding as implemented in DFTB+ version 19.1113. All atom pairs were described using standard parameters from the mio-0-1 parameter set114. Lattice parameters were optimized at the same time as atomic coordinates.Geometries of monolayer, AA, AB and slip-stacked TTA-SPDFP COF and TTA-MCDFP COFs with 33, 66 and 100% functionalized linkers were built from fragments using AuToGraFS115. The TTA-SPDFP and TTA-MCDFP COF structures were constructed from the CIF files, A 6x6 layer of 6 stacked sheets were placed in a vacuum slab of z = 9.5 nm. The simulation box along the x, y-axis adjusts such that PBC extends the simulation dimensions to higher length scales. The structures were then placed in a simulation box of dimensions 19.6 × 16.8 nm and solvated with water molecules, resulting in approximately 180,000 particles in each simulation box. The DREIDING force field116 was used to represent the COFs, while the TIP3P model117 was used to represent water. This parameter set has been previously shown to give good agreement with experimental data in our previous studies118.All-atom molecular dynamics simulations were performed using the LAMMPS software–5 kJ/mol119. A time step of 2 fs was used, and simulations were run for approximately 25 ns for aqueous systems, with monitoring of convergence of the structural ensemble. The last 8 ns of the simulation were used for analysis. For vacuum simulations, the simulations were extended up to 100 ns, with the last 10 ns used for data analysis. The radial distribution functions were computed using the gmx_rdf module of GROMACS, and the bending angle was computed using the gmx_gangle module of GROMACS120. The membrane curvature was computed using the MDAnalysis Membrane Curvature tool which calculates the Gaussian curvature averaged over the frames of the last 3 ns of the trajectory.We investigated the behavior of the TTA-SPDFP and TTA-MCDFP COF structures in both vacuum and aqueous environments. For each COF system, the initial configuration was optimized by geometry optimization using the steepest descent for 5000 steps, allowing the system to reach its minimum energy. Then, we conducted MD simulations in an NPT ensemble, with the temperature set to 298.15 K and pressure set to 1 atm. Periodic boundary conditions were applied in all directions, allowing for volume and shape changes during the MD simulations. Pairwise interactions were truncated at a cutoff of 10 Å, and Ewald summation was employed for long-range Coulombic interactions with an accuracy of 1 × 105 and the reaction mixture heated at 90 °C for three hours. After the reaction completion, the reaction mixture was cooled to room temperature and diluted the reaction mixture with 60 mL of anhydrous chloroform. Then, the reaction mixture was cooled to 0 °C and 25 mL of methanol was added dropwise under stirring. After the addition of excess methanol to the reaction mixture, it was left to stir for an additional 30 min. The reaction mixture was filtered, washed with methanol and then dried. The product 2a obtained as a white solid . 1H NMR : δ 4.02 , 8.45 ; 13C NMR : δ 53.4, 131.2, 135.1, 149.2, 164.1; MS (ESI-HRMS): m/z Calcd for (C9H9BrNO4)+: 273.9709 (M+H)+, found: 273.9711 (M+H)+.5.0 g of chelidamic acid hydrate was stirred with 25.0 g of PBr4 added in portions over a period of 15 min to the reaction mixture. Then, the reaction mixture was refluxed for 24 h, and allowed to cool to room temperature after the completion the reaction. The reaction mixture was extracted with ethyl acetate (3 x 30 mL). The organic phase was collected and dried over sodium sulphate and evaporated to dryness. The compound 2b was obtained in 62% (3.14 g) yield. 1H NMR : δ 4.51 , 7.52 ; 13C NMR : δ 64.1, 121.6, 133.9, 163.4; MS (ESI-HRMS): m/z Calcd for (C7H9BrNO2)+: 217.9811 (M+H)+, found: 217.9812 (M+H)+.6.39 g of dimethyl-4-bromopyridine-2,6-dicarboxylate in 125 mL absolute ethanol was stirred in the 250 ml round bottom flask and 4.25 g of NaBH2 in 25 mL 1,4-dioxane under reflux condition for 20 h. Then, the reaction mixture was filtered through Celite and solvent was removed under reduced pressure. The product was purified by silica gel column using dichloromethane as an eluent. The product 2c was obtained as a pink solid in 69% yield (0.97 g) 1H NMR : δ 8.31 , 10.14 ; 13C NMR : δ 128.8, 136.0, 154.0, 191.4; MS (ESI-HRMS): m/z Calcd for (C7H5BrNO2)+: 213.9498 (M+H)+, found: 213.9499 (M+H)+.1.43 g of 4-bromo-2,6-pyridinedimethanol was added 0.73 g of SeO121. 5-bromo-2-hydroxybenzaldehyde , bis(pinacolato)diboron , potassium acetate , and ferrocene)-dichloropalladium(II)  in 10 mL of 1,4-dioxane were added to a 50 mL microwave reaction vessel. The reaction mixture was refluxed under microwave irradiation for 15 min. After completion of the reaction, dioxane was removed and the residue was diluted with 20 mL of water and then extracted with dichloromethane (3 × 15 mL). The combined organic layers were dried under anhydrous magnesium sulphate, filtered, and then the solvent was removed under reduced pressure. The obtained crude product was purified using combi flash column chromatography with a hexane and ethyl acetate (10:1) gradient. Compound 4a was obtained in 87% (0.53 g) yield. 1H-NMR  δ 1.37 , 6.99 , 7.96 , 8.07 , 9.94 , 11.24 ; 13C NMR : δ 24.9, 84.08, 117.1, 120.4, 141.4, 143.2, 164.0, 196.4; MS (ESI-HRMS): m/z Calcd for (C13H18BO4)+: 249.1292 (M+H)+, found: 249.1289 (M+H)+.Compound 4a was prepared according to the previously reported synthetic procedure with slight modification3N . The reaction mixture was refluxed under argon for 3 h. After the completion of the reaction, the solvent was removed under reduced pressure. The obtained solid was purified using combi flash chromatography using hexane and ethyl acetate (1:19) gradient. Compound 4b was obtained in 71% (0.572 g) yield as pale pink solid. 1H-NMR  δ 1.19 , 1.31 , 1.35 , 2.74 , 5.67 , 6.53 , 6.72 , 6.84-6.91 , 7.08 , 7.18-7.21 , 7.55 , 7.58 ; 13C NMR : δ 20.0, 24.8, 25.9, 28.9, 51.7, 83.6, 104.6, 106.8, 114.6, 118.3, 119.0, 119.2, 121.5, 127.6, 129.5, 133.6, 136.8, 148.2, 157.3; MS (ESI-HRMS): m/z Calcd for (C25H31BNO3)+: 404.2391 (M+H)+, found: 404.2389 (M+H)+.0.500 g (2.0 mmol) of compound 4a and 1,2,3,3-tetramethyl-3H-indol-1-ium iodide  were dissolved in freshly distilled EtOH (10 mL) and anhydrous Et3)4  were added to a 100 mL three neck round-bottom flask under argon atmosphere. 20 mL of 1:1 THF/H2O mixed solvent containing Na2CO3 (20 % solution) degassed solution was added to the reaction mixture. Then, the reaction mixture was refluxed at 85 °C for 20 h under an inert argon atmosphere. After completion of the reaction, 30 mL of distilled water was added to the reaction mixture and the aqueous solution was extracted with dichloromethane (3 × 25 mL). The collected organic layer was dried under anhydrous magnesium sulphate for 30 min. The resulting solution was filtered and concentrated to dryness. The resulting residue was purified by flash column chromatography [SiO2: Hexane and CH2Cl2 (1:4)] to afford SPDFP as a light green color solid in 0.427 g (56%) yield. 1H NMR : δ 1.22 , 1.35 , 2.79 , 5.83 , 6.57 , 6.88-6.91 , 6.97 , 7.11 , 7.21-7.24 , 7.52 , 7.55 , 8.36 , 10.23 ; 13C NMR : δ 20.1, 25.9, 28.9, 52.0, 105.2, 106.9, 116.2, 119.4, 119.7, 120.9, 121.5, 121.9, 125.4, 127.5, 127.7, 128.6, 128.8, 136.4, 148.0, 150.4, 153.6, 156.7, 192.7; MS (ESI-HRMS): m/z Calcd for (C26H23N2O3)+: 411.1704 (M+H)+, found: 411.1707 (M+H)+.Compound 4b , 4-bromo-2,6-diformylpyridine (2c)  and Pd(PPh122. 1.54 g (13.08 mmol) of 4-aminobenzonitrile in a 50 mL oven-dry round bottom flask was stirred at 0 °C under an argon atmosphere and 4 mL of trifluoromethanesulfonic acid was added in dropwise for 10 min to the reaction mixture, maintaining the reaction temperature at 0 °C. Then, the reaction mixture was stirred at room temperature for 24 h in an argon atmosphere. After the reaction completion, 40 mL distilled water added to the reaction mixture and neutralized it by adding 2M NaOH solution until the pH reaches to 7. The obtained pale-yellow precipitate was filtered, wash several times with distilled water and then dried under vacuum. The pale-yellow solid of TTA obtained in 78 % yield. 1H NMR : δ 5.91 , 6.69 , 8.35 ; 13C NMR : δ 113.6, 123.4, 130.6, 153.4, 170.1; MS (ESI-HRMS): m/z Calcd for (C21H19N6)+: 355.1665 (M+H)+, found: 355.1667 (M+H)+.TTA was synthesized according to previously reported methods109. 2,6-pyridinedimethanol , and selenium dioxide  in dioxane (100 mL) and water (4%) in a 250-mL round-bottom flask was refluxed for 20 h. After the completion of the reaction, the mixture was filtered in hot condition through celite bed. The filter was washed with hot dioxane (2 x 10 mL) and CH2Cl2 (2 x 10 mL). Solvents were removed from the filtrate under reduced pressure and the obtained solid was purified by silica gel column using CH2Cl2 as an eluent to afford DFP as a pale pink solid. Yield: 4.0 g, 76%. 1H NMR : δ 8.09 , 8.17 , 10.16 ; 13C NMR : δ 125.5, 138.6, 153.2, 192.6; MS (ESI-HRMS): m/z Calcd for (C7H6NO2)+: 136.0393 (M+H)+, found: 136.0395 (M+H)+.DFP was synthesized by following the previously reported synthetic procedureThe spiropyran-functionalized COF (TTA-SPDFP) was prepared via imine condensation of the monomers 4,4′,4″--trianiline , pyridine-2,6-dicarbaldehyde , and (R)-4--pyridine-2,6-dicarbaldehyde . The reactions were run in 1,4-dioxane using acetic acid  as a catalyst at room temperature under sonication in a 25 mL glass vessel at room temperature for 2 h. The resulting product was obtained as a yellow powder. The product was purified by washing with ethanol, followed by drying at 120 °C for 12 h. The product was completely insoluble in water and common organic solvents. Sonochemical reactions were performed with a 550 W ultrasonic probe in continuous mode using a 3 mm microtip probe at 40% sonication power (the maximum power recommended for the used probe).Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


The Zn/HA-ML coating showed a sixfold decrease in the corrosion current density and more improvements in the corrosion resistance by twofold after an immersion time of 14 days, which was mainly attributed to newly formed apatite and corrosion by-products of Zn particles. The Zn/HA-ML coating effectively combined the advantages of the corrosion resistance of CS Zn underlayer and the bioactivity of HVSFS Zn/HA multi-layers, which proposed a low-temperature strategy for improving corrosion resistance and bioactivity for implant metals.The study aims to enhance the corrosion resistance and bioactivity of Mg alloy substrates through the development of a zinc/hydroxyapatite multi-layer (Zn/HA-ML) coating. The Zn/HA-ML coating was prepared by depositing a cold-sprayed (CS) Zn underlayer and a high-velocity suspension flame sprayed (HVSFS) Zn/HA multi-layer and was compared with the CS Zn coating and the Zn/HA dual-layer (Zn/HA-DL) coating. Phase, microstructure, and bonding strength were examined, respectively, by X-ray diffraction, scanning electron microscopy, and tensile bonding testing. Corrosion behavior and bioactivity were investigated using potentiodynamic polarization, electrochemical impedance spectroscopy, and immersion testing. Results show that the HVSFS Zn/HA composite layers were mainly composed of Zn, HA, and ZnO and were well bonded to the substrate. The HVSFS HA upper layer on the CS Zn underlayer in the Zn/HA-DL coating exhibited microcracks due to their mismatched thermal expansion coefficient (CTE). The Zn/HA-ML coating exhibited good bonding within different layers and showed a higher bonding strength of 27.3 ± 2.3 MPa than the Zn/HA-DL coating of 20.4 ± 2.7 MPa. The CS Zn coating, Zn/HA-DL coating, and Zn/HA-ML coating decreased the corrosion current density of the Mg alloy substrate by around two–fourfold from 3.12 ± 0.75 mA/cm Magnesium alloys exhibit some advantages of biodegradability, biocompatibility, and proper mechanical properties comparable to the human bone. However, rapid degradation rates limit their mechanical stabilities and their wider application in the field of implant materials . In addiFunctionally graded materials (FGMs) are innovative materials with compositions and/or microstructures gradually varying in space, which is a promising strategy for bio-metal substrates ,6. GradeFunctionally graded coatings or multi-layer coatings are successfully prepared by different technologies ,16,17,182O3 composite coating [2O3 composite coatings [Cold spray is an excellent technology for preparing metal coatings, which depends on the high-velocity impact of metal particles on the metal surface . Cold-sp coating . The 20  coating , Al/A2O3coatings , and Zn/coatings . TherefoHA-based coatings are usually prepared by different technologies, such as plasma spraying , sol-gelIn the present study, a Zn/HA-ML coating was prepared on the AZ31B Mg alloy substrate, which was composed of a CS Zn underlayer and a HVSFS Zn/HA multi-layer. Phase, microstructure, bonding strength, corrosion behavior, and bioactivity of the Zn/HA-ML coating were investigated by comparing with the CS Zn coating and the Zn/HA dual-layer coating.2 primary gas pressure and temperature were 2.0 ± 0.1 MPa and 260 ± 20 °C, respectively. The spray distance was fixed to 20 mm, and the transverse speed of the torch was 50 mm/s.Zn powder  was used as the original material for Zn coating. A mixed powder of 50 vol.% Zn powder and 50 vol.% 1Cr18 shots  was used for cold spraying, as shown in Nano-size HA powder  and Zn powder were the original materials for Zn/HA dual-layer coating and Zn/HA multi-layer coating. Three types of mixed particle suspensions  were prepared by mixing HA powders with Zn powders at the weight proportions of 10, 3:7, and 7:3, respectively. Different mixed particle suspensions were prepared by dispersing 10 wt.% of pure HA powders or Zn/HA mixed powders into 90 wt.% of solvent. The solvent was composed of distilled water and ethanol in a 1:1 weight proportion. Different particle suspensions were fully mixed for at least 10 h by a mechanical stirrer.An HVSFS system was applied to deposit the pure HA layer and the Zn/HA composite layers, as shown in Kα radiation of 1.5418 Å, operating voltage of 35 kV, operating current of 35 mA, 2θ range of 20–90°, and a scan rate of 0.1 °/s. Surface morphology and cross-sections of CS Zn coating, Zn/HA-DL coating, and multi-layer coating, as well as after immersion, were investigated by scanning electron microscopy  with energy-dispersive spectrometry (EDS). Phase compositions of the Zn/HA-ML coating before and after immersion were examined by Fourier-transform infrared spectroscopy  using KBr pellets technology with a scan range of 400–4000 cm−1.Phase structures of different coatings were identified by X-ray diffraction  at CuTo investigate the bonding strength of the CS Zn coating, the Zn/HA-DL coating, and the Zn/HA-ML coating, a standard tensile test method (ASTM C633)  was appl2 were exposed to simulated body fluid , and other surfaces were covered by epoxy resin. The SBF contained 142.0 mM Na+, 5.0 mM K+, 1.5 mM Mg2+, 2.5 mM Ca2+, 103.0 mM Cl−, 10.0 m HCO3−, 1.0 mM HPO42−, and 0.5 mM SO42−. During immersion, the volume of SBF followed the equation Vs = Sa/10 [s. is the volume of SBF (mL) and Sa is the apparent surface area of the specimen (mm2). During electrochemical testing, a flat three-electrode cell consisting of a working electrode (coating or bare substrate), a reference electrode (Ag/AgCl electrode), and a counter electrode (Pt plate) was utilized. Polarization curves were carried out on an electrochemical workstation  at open circuit potential from −0.5 V to 0.5 V (vs. SCE) with a scan rate of 1 mV/s. CorrWare software  was employed to estimate the corrosion potential (Ecorr), corrosion current density (Icorr), and corrosion rate. EIS measurements were carried out at open-circuit potential (OCP) with an amplitude of 10 mV and with a scanning frequency from 0.01 Hz to 100 kHz. Recorded impedance spectra were fitted by using Z-view software .To evaluate the corrosion behaviors of different coatings, potentiodynamic polarization testing and electrochemical impedance spectroscopy (EIS) were applied. Before electrochemical testing, different coatings were immersed in simulated body fluid (SBF) at 37 ± 1 °C for 1, 7, and 14 days. During immersion testing, different coatings on the substrate with an area of 1 cm = Sa/10 , where v2 could enhance the HA decomposition [position , but theposition . The phaposition . This re−6/°C [−6/°C [−6/°C [−6/°C , 30 × 10C [−6/°C , and 10.C [−6/°C , respectIn the Zn/HA-ML coating, the CS Zn underlayer, the HVSFS Zn/HA composite layers, and the pure HA upper layer were obviously differentiated, as shown in Tensile strengths of the CS Zn coating, Zn/HA-DL coating, and Zn/HA-ML coating were measured as shown in Fractured surfaces of different coatings were examined, as shown in Ecorr), corrosion current density (Icorr), and corrosion rate. The Tafel slopes are listed in Icorr × K × EW)/(d × A), in which Icorr is corrosion current, K is constant, EW is equivalent weight, d is density, and A is area. It can be seen that the CS Zn coating, Zn/HA-DL coating, and Zn/HA-ML coating exhibited higher Ecorr, lower Icorr, and lower corrosion rates than the bare Mg alloy substrate after immersion of 1 day. Compared to the bare substrate, the decrease in both Icorr and corrosion rate of different coatings was around threefold and twofold, respectively. It indicates the prior corrosion resistance of different coatings could be attributed to the high anti-corrosion of Zn coating [Icorr from 1.41 ± 0.82mA/cm2 to 1.06 ± 0.31 mA/cm2 and 0.88 ± 0.27 mA/cm2 and decreased the corrosion rate from 16.5 ± 3.6 mm/year to 12.5 ± 3.7 mm/year and 10.3 ± 3.2 mm/year, respectively. This result confirms that the HVSFS HA upper layer and the HVSFS Zn/HA composite layers improved corrosion resistances of the CS Zn underlayer, which could be mainly related to the lower conductivity of HA.To examine the corrosion behaviors of different coatings, potentiodynamic polarization was measured after different immersion times.  coating . CompareIcorr and corrosion rates, although the corrosion potentials were stable. Compared with an immersion time of 1 day, the CS Zn coating after an immersion time of 14 days exhibited a decrease in Icorr and corrosion rate by around twofold from 1.41 ± 0.82 mA/cm2 to 0.73 ± 0.12 mA/cm2 and from 16.5 ± 3.6 mm/year to 8.5 ± 1.2 mm/year, respectively. The Zn/HA-DL coating after an immersion time of 14 days exhibited a decrease in Icorr and corrosion rate by around threefold from 1.06 ± 0.31 mA/cm2 to 0.37 ± 0.12 mA/cm2 and from 12.5 ± 3.7 mm/year to 4.4 ± 1.5 mm/year, respectively. The Zn/HA-ML coating after an immersion time of 14 days exhibited a decrease in Icorr and corrosion rate by around sixfold from 0.88 ± 0.27 mA/cm2 to 0.14 ± 0.07 mA/cm2 and from 10.3 ± 3.2 mm/year to 1.7 ± 0.9 mm/year, respectively. The improvement in anti-corrosion properties after immersion could be mainly attributed to the formation of corrosion byproducts [By prolonging the immersion time to 7 days and 14 days, the CS Zn coating, Zn/HA-DL coating, and Zn/HA-ML coating exhibited lower products . This reRs represents the ohmic resistance of the substrate and conductive lines, and R1 and CPE1 represent the charge transfer resistance and capacitive characteristics at the bare substrate/electrolyte interface. For different coatings, according to the literature [R1 and CPE1 represent the charge transfer resistance and capacitive characteristics at the dense coating/electrolyte interface, respectively; R2 and CPE2 represent the charge transfer resistance and capacitive of the porous Zn/HA composite layer/electrolyte interface or HA/electrolyte interface, respectively. Fitting results of EIS for different coatings are listed in To further investigate the corrosion behaviors of different coatings, the EIS was measured and is shown in terature ,22,23, RRp = R1 + R2) [Rp) of the CS Zn coating, the Zn/HA-DL coating, and the Zn/HA-ML coating after an immersion time of 1 day were higher than that of the bare substrate by around sixfold, sixteenfold, and elevenfold, respectively. With the extension of immersion time to 14 days, the Rp of all the coatings was further increased by around one–threefold. It can be seen that the Rp of the Zn/HA-DL coating and the Zn/HA-ML coating was higher than the CS Zn coating after different immersion periods. This result indicates that the HVSFS HA upper and the Zn/HA composite layers contributed to the improvement in corrosion resistance of the CS Zn coating.It can be found that all the coatings exhibited higher corrosion resistance than the bare substrate and improvements in corrosion resistance after immersion, which was consistent with the results of polarization properties. Compared to the bare Mg alloy substrate, all the coatings exhibited higher polarization resistances (R1 + R2) . PolarizIt is reported that the cold-sprayed Zn/HA  and Ti/HAfter immersion for 14 days, the CS Zn coating was covered by some clusters with tiny particles (yellow arrows in 43− and OH− were the main functional groups, as shown in 2 to 1.41 ± 0.82mA/cm2, 1.06 ± 0.31 mA/cm2, and 0.88 ± 0.27 mA/cm2, respectively, and exhibited significant decreases in corrosion current density to 0.73 ± 0.12 mA/cm2, 0.37 ± 0.12 mA/cm2, and 0.14 ± 0.07 mA/cm2 after an immersion time of 14 days. The Zn/HA-ML coating after an immersion time of 14 days showed more decreases in the corrosion current density by sixfold and more improvements in the corrosion resistance by twofold, which was mainly attributed to newly formed apatite and corrosion by-products of Zn coatings on/within the coating. This Zn/HA-ML coating effectively combined the advantages of the corrosion resistance of the CS Zn layer and bioactivities of the HVSFS Zn/HA composite layers, which proposed a low-temperature strategy for improving corrosion resistance and bioactivities.In this study, a Zn/HA-ML coating was prepared on a Mg alloy substrate by combining cold spraying and high-velocity suspension flame spraying. HVSFS Zn/HA composite layers were mainly composed of Zn and HA in addition to ZnO, and the HVSFS HA upper layer was not decomposed. The HVSFS HA upper layer in the Zn/HA-DL coating presented microcracks due to the CTE mismatch. The Zn/HA-ML coating effectively decreased the CTE mismatch and showed a higher bonding strength of 27.3 ± 2.3 MPa than the Zn/HA-DL coating of 20.4 ± 2.7 MPa by 30%. Both the CS Zn coating, the Zn/HA-DL coating, and the Zn/HA-ML coating decreased corrosion current density of the Mg alloy substrate by around two–fourfold from 3.12 ± 0.75 mA/cm