As digital medicine has exerted profound influences upon diagnosis and treatment of hepatobiliary diseases, our study aims to investigate the accuracy of three-dimensional visualization and evaluation (3DVE) system in assessing the resectability of hilar cholangiocarcinoma (hCCA), and explores its potential clinical value.The discovery cohort, containing 111 patients from April 2013 to December 2019, was retrospectively included to determine resectability according to revised criteria for unresectability of hCCA. 3D visualization models were reconstructed to evaluate resectability parameters including biliary infiltration, vascular involvement, hepatic atrophy and metastasis. Evaluation accuracy were compared between contrast-enhanced CT and 3DVE. Logistic analysis was performed to identify independent risk factors of R0 resection. A new comprehensive 3DVE classification of hCCA based on factors influencing resectability was proposed to investigate its role in predicting R0 resection and prognosis. The main outcomes were also analyzed in cohort validation, including 34 patients from January 2020 to August 2022.p = 0.03). By multivariable analysis, hepatic artery involvement in 3DVE was identified an independent risk factor for R1 or R2 resection . New 3DVE hCCA classification was valuable in predicting patients’ R0 resection rate (p < 0.001) and prognosis (p < 0.0001). The main outcomes were internally validated.3DVE showed an accuracy rate of 91% (95%CI 83.6–95.4%) in preoperatively evaluating hCCA resectability, significantly higher than 81% (95%CI 72.8–87.7%) of that of CT (3DVE exhibited a better efficacy in evaluating hCCA resectability, compared with contrast-enhanced CT. Preoperative 3DVE demonstrated hepatic artery involvement was an independent risk factor for the absence of R0 margin. 3DVE classification of hCCA was valuable in clinical practice.The online version contains supplementary material available at 10.1186/s12957-023-03126-2. Hilar cholangiocarcinoma (hCCA), arising from the common hepatic duct or first-class hepatic ducts, is a biliary malignancy with an incidence less than 1 per 100,000 men in the most countries . As prevTo assess biliary infiltration, endoscopic retrograde cholangiography (ERC), percutaneous transhepatic cholangiography (PTC), and magnetic resonance cholangiopancreatography (MRCP) yielded accuracy rates of 87%, 40–90%, and 80–95%, respectively, which are superior to 80% of contrast-enhanced computed tomography (CT) . WhereasThree-dimensional visualization and evaluation (3DVE) system is a digital image processing technology used for hepatobiliary diseases –17. 3DVEHere, we explore the efficacy of 3DVE in assessing hCCA resectability by comparing with that of CT. In addition, we proposed a new 3DVE classification of hCCA and explored its clinical value in predicting surgical outcomes and prognosis.The study flowchart was shown in Fig. The institutional electronic patient database was searched for patients pathologically confirmed hCCA, with the following inclusion criteria: (a) underwent preoperative contrast-enhanced CT and 3DVE; (b) underwent full surgical exploration.All the patients held their breath in full inspiration before scanning and were instructed to do so during each scan phase. A preoperative triple-phase CT was taken using a 64-slice CT scanner  following the standard protocol that involved a slice thickness for axial images of 1.25 mm, a reconstruction slice thickness of 1.25 mm, and a reconstruction interval of 1.25 mm. After administration of nonionic iodinated contrast material  with an injection rate of 3–4 mL/s, the arterial and portal venous phase scans were acquired in 20–25 s and 60–65 s, respectively. The contrast-enhanced CT data were sent to the picture archiving and communication system (PACS) for interpretation.CT data in DICOM format were transferred to an IQQA-LIVER workstation . The 3D visualization processing was structured as a five-step procedure. Firstly, imported contrast-enhanced CT data were automatically processed into an original 3D liver model. Secondly, the liver contour was constructed in portal venous phase by the semi-automated seeded region growing segmentation technique and the active contour was manually modified. Thirdly, the HA was automatically extracted based on the contrast agent in the arterial phase and PV and hepatic veins were extracted from the portal venous phase. Dilated bile ducts, seen as hypodensity structures in the portal venous phase, were extracted by reverse processing, and 3D bile duct images were rendered. The course, morphology and continuity of blood vessels and biliary tract were checked, and manual revision was necessary if branches were wrongly reconstructed. Then, the vasculature structures from two phases were registered and merged. Fourthly, a seed point was selected in the center of the tumor and the borders of the tumor were manually outlined in coronal, sagittal, and horizontal planes, respectively Figure E. FinallyAll contrast-enhanced CT images were retrospectively reviewed in consensus with a hepatopancreatobiliary surgeon (W.C) and an abdominal radiologist (L.J.Q). The radiologists reviewed the CT images on the PACS. The three-dimensional visualization evaluation (3DVE) was collaboratively accomplished by two hepatopancreatobiliary surgeons  and an abdominal radiologist (J.Z). The 3D visualization images were viewed and assessed on the IQQA-LIVER workstation. All observers were aware of the diagnosis of hCCA but were blinded to other clinicopathological features of the patients, the agreement value  of observers were calculated. After the first image analysis, an experienced doctor  reviewed the discordant findings between observers to decide which result to use.The following imaging features were qualitatively analyzed for resectability evaluation. Biliary infiltration was considered present if (a) the ductal wall was hyperattenuating compared with the liver in the portal venous phase, (b) if there was irregular ductal wall thickening with an asymmetric upstream intrahepatic ductal dilation, or (c) if the lumen was obliterated by intraductal soft-tissue , 23. TheWe revised the criteria for the unresectability of hCCA by combining the criteria proposed by Jarnagin  and Lee Liver resection procedures mainly involved minor hepatectomy, hemihepatectomy, trisegmentectomy, and specific hepatectomy. Lymphadenectomy was performed for clearance of all the lymph nodes of the 8th, 12th, and 13th groups. The operative approach of major hepatectomy referred to previous studies , 29. Tajχ2 test, and continuous variables were compared using an unpaired two-sided t test. The associations between ordinal categorical variables and dichotomous variables were analyzed using the Cochran-Armitage trend test. Cohen’s Kappa analysis was calculated, and κ values < 0 indicated no agreement and 0 < κ ≤ 0.2 slight, 0.2 < κ ≤ 0.4 fair, 0.4 < κ ≤ 0.6 moderate, 0.6 < κ ≤ 0.8 substantial, and 0.8 < κ ≤ 1 were almost perfect agreement. Ordinal categorical variables were compared using the Spearman rank correlation analysis. Multivariable logistic regression analysis was performed using the backward likelihood ratio method, including all variables with p < 0.1 in univariate analysis. P < 0.05 was considered statistically significant. A Kaplan–Meier curve was generated and Logrank test was performed for survival analysis. The statistical analysis was conducted using the SPSS version 19.0 software  and GraphPad Prism version 8.0.2.Categorical variables were compared using the n = 66); (b) underwent neoadjuvant therapy before surgery (n = 66); (c) an incomplete pathological examination that influenced the adjudgment of resectability (n = 38). The discovery cohort contained 111 patients from April 2013 to December 2019, the other 34 patients from January 2020 to August 2022 formed validation cohort.Of 315 eligible patients from April 2013 to August 2022, 170 were excluded due to one of the following reasons: (a) an interval greater than 6 weeks between CT imaging and surgery, and poor CT quality (n = 10), bilateral tumor extension to the limits of hepatic duct (n = 1), one hepatic lobe with contralateral vascular invasion (n = 1), tumor extension to unilateral dissection limit of bile duct and contralateral vascular invasion (n = 5), metastasis to periaortic, pericaval, superior mesenteric artery, or celiac lymph nodes (n = 9), distant metastasis (n = 4) and insufficient FLR volume (n = 1). According to the surgical findings, curative-intent surgery was attempted in 14 patients, and 17 patients were unresectable due to invasion of the main PV or proper HA (n = 12), bilateral tumor extension to the limits of hepatic duct (n = 2), distant metastasis (n = 1), and insufficient FLR volume (n = 2). According to revised unresectability criteria for hCCA, we found that HA involvement, PV involvement, hepatic lobar atrophy, lymph node metastasis and distant metastasis were more frequently observed in 3DVE unresectable group (P < 0.05)  with a mean age of 65 years ± 10 . The clinical classification is shown in Table EThe previously mentioned surgical resectability factors were respectively evaluated by contrast-enhanced CT Fig.  and 3DVEp = 0.033). The agreement value between 3DVE and intraoperative findings with pathological examinations achieved 0.762, higher than that of 0.507 of contrast-enhanced CT , and 25 patients were unresectable. By contrast-enhanced CT, 79 patients were allocated to resectable group and 32 to the unresectable group. According to preoperative 3DVE, 80 patients were resectable and 31 patients belonged to the unresectable group. Compared with CT, 3DVE showed greater sensitivity , specificity , positive predictive value (PPV) , negative predictive value (NPV) , and overall accuracy  and lymph node metastasis  were significantly associated with the absence of R0 resection. The multivariable logistic regression analysis showed that the R0 resection rate of patients without HA involvement in preoperative 3DVE was 81.4% , which is significantly higher than 52.9%  of the patients with HA involvement. Ultimately, we identified HA involvement as an independent risk factor associated with R1 or R2 resection  . The R0 resection rates of hCCA of MSKCC T1, T2, and T3 were 73.5%, 69.0%, and 46.9%, respectively (p < 0.05) (26). The R0 resection rates based on Bismuth-Corlette classification and AJCC TNM staging system (8th edition) were not statistically significant . For the Bismuth-Corlette system, the 1-year survival rates were 94.1%, 79.8%, 70.5%, and 40.4%, respectively, for type I, II, III, and IV, with corresponding median OS of 45 months,43 months, 23 months, and 9 months (p < 0.01). Based on the MSKCC T staging system, the 1-year survival rates were 88.9%, 71.9%, and 51.1%, respectively, for T1, T2, and T3, with corresponding median OS of 43 months, 23 months, and 13 months (p < 0.01). In the 8th AJCC TNM staging system, the 1-year survival rate were 66.7%, 92.8%, 73.3%, and 21.3%, respectively, for hCCA patients of TNM I, II, III, and IV, with corresponding median OS of 43 months, 43 months, 24 months, and 9 months (p < 0.001) . The R0 resection rate was 70.8% in the group without HA involvement, which was significantly higher than 20.0% of HA involvement group (P = 0.004)  with a mean age of 63 years ± 11 . The baseline information is shown in Table EP < 0.05), sensitivity, specificity, NPV, and PPV in evaluating hCCA resectability, compared with those of CT. Specifically, 3DVE exhibited superiority in evaluating biliary infiltration, HA invasion, and hepatic atrophy of hCCA, compared with CT. It also performed similarly to CT in assessing P (U) point involvement and metastasis.A preoperative radiological examination is fundamental for the evaluation of surgical resectability and the ultimately selection of patients with a high probability of R0 resection. MRCP, PTC and ERCP could well assess the biliary infiltration , 11, 12.Through 3D reconstruction, stereoscopic and intuitive visual models of biliary tree and blood vessels were rendered. 3DVE enhanced the comprehension of continuity and course of vasculature system, the individualized biliary tree, and the variants and origin of HA . HoweverBecause 3D biliary tree image was extracted in portal venous phase, the P point and U point could be precisely located in reference to the course of portal vein, which facilitates the identification of the important landmarks for surgical planning Figure E 17, 28], 2817, 2R0 margin was a favorable prognostic factor for hCCA , 5. Our Several classification systems were widely utilized for predicting prognosis and R0 resection rate of hCCA patients –5. HowevBesides preoperative diagnosis, Fang et al.  and ZhanOur study had some limitations. First, it is a single-center, retrospective study with inherent biases. Multicentral data could be included and compared, since a prospective study may further evaluate the efficacy of the 3DVE system. Second, the 3D reconstruction was the secondary processing of images originating from raw images, and thus, the 3D reconstruction could not provide additional information. The quality of 3D visualization models was also influenced by the experience of the manipulators. Third, as slight bile duct dilation was not clearly differentiated in CT, 3D reconstruction might fail to apply to hCCA without an obvious bile duct dilatation. Fourth, the multiple variables analysis was based on the 3DVE, and not all the odds rates of variables were statistically significant, so we did not formulate a classification entirely based on the odds rates or even determined each stage. Thus, our classification was formulated based on combination of the canonical hCCA classification and clinical practice.In conclusion, 3DVE exhibited a significantly higher accuracy in the preoperative evaluation of hCCA resectability in comparison to contrast-enhanced CT. The tumor involvement of HA in 3DVE was identified as the independent risk factor for the absence of R0 margin of hCCA. Additionally, the proposed preoperative 3DVE classification system performed well in predicting R0 resection rate and prognosis of hCCA patients.Additional file 1:Table E1. Result of clinical classification of 111 patients of hilar cholangiocarcinoma. Table E2. Resection radicality according to surgical procedures of 111 patients of hilar cholangiocarcinoma. Table E3. Evaluation of tumor longitudinal infiltration by CT/3DVE and intraoperative findings combined with pathological examination. Table E4. CT evaluation of resectability factors in hilar cholangiocarcinoma. Table E5. 3D visualization and evaluation of resectability factors in hilar cholangiocarcinoma. Table E6. Contents of classification systems of hilar cholangiocarcinoma. Table E7. Clinical 3DVE classification of hilar cholangiocarcinoma. Table E8. Percentage of R0 resection according to different staging systems of 111 patients of hilar cholangiocarcinoma. Table E9. Clinicopathological features of validation cohort. Table E10. Resectability evaluated by CT and 3DVE in validation cohort. Table E11. R0 resection rate in validation cohort.Additional file 2:Figure E1. Restoration of 3D structure of lesions of hilar cholangiocarcinoma and adjustment of tumor border in 2D images. Figure E2. Simulation of resection of right anterior lobe of the liver and resection effect. Figure E3. 3D visualization model of hilar cholangiocarcinoma according to Bismuth-Corlette classification. Figure E4. 3D visualization models to measure the distance between P and U points and tumor border. Figure E5. Actual reconstruction diagrams of 3DVE classification of hilar cholangiocarcinoma.


CHRNA5 mRNA is upregulated in several types of cancer, including lung, prostate, colorectal, and stomach cancer, and cancer severity is correlated with smoking. In this study, we investigate the contribution of α5 in the nicotine-induced cancer hallmark functions proliferation and migration, in breast, colon, and prostate cancer cells. Nine human cell lines from different origins were used to determine nAChR subunit expression levels. Then, selected breast (MCF7), colon (SW480), and prostate (DU145) cancer cell lines were used to investigate the nicotine-induced effects mediated by α5. Using pharmacological and siRNA-based experiments, we show that α5 is essential for nicotine-induced proliferation and migration. Additionally, upon downregulation of α5, nicotine-promoted expression of EMT markers and immune regulatory proteins was impaired. Moreover, the α5 polymorphism D398N (α5SNP) caused a basal increase in proliferation and migration in the DU145 cell line, and the effect was mediated through G-protein signaling. Taken together, our results indicate that nicotine-induced cancer cell proliferation and migration are mediated via α5, adding to the characterization of α5 as a putative therapeutical target.Nicotine in tobacco is known to induce tumor-promoting effects and cause chemotherapy resistance through the activation of nicotinic acetylcholine receptors (nAChRs). Many studies have associated the α5 nicotinic receptor subunit (α5), and a specific polymorphism in this subunit, with (i) nicotine administration, (ii) nicotine dependence, and (iii) lung cancer. The α5 gene  In + ions ,22+) ions . Similar2+) ions ,6, nAChR2+) ions ,8,9. Alt2+) ions , they ar2+) ions ,12. It i2+) ions ,14,15. E2+) ions ,17.2+ influx, epithelial to mesenchymal transition (EMT), and to initiate specific signaling cascades because of nicotine’s interaction with nAChRs [2+ permeability, such as in the α4β2α5 and α3β4α5 receptors [In the context of lung cancer, nicotine exposure is known to increase cell proliferation, cell invasion, migration, Cah nAChRs ,21,22,23h nAChRs ,27,28,29h nAChRs ,31,32. Ah nAChRs ,34,35. Ih nAChRs ,37,38, weceptors . To dateeceptors . Notablyeceptors ,42 and ieceptors . Consequo–protein interactions [Further studies have shown that a specific polymorphism in this subunit, which changes an aspartate, D, in position 398 to an asparagine, N, (referred to as α5SNP), is strongly linked with nicotine dependence, quantity of smoking, and lung cancer ,45,46,47ractions  but withractions . Likewisractions .Apart from lung cancer, current data also associate cigarette smoking with other types of cancer, such as prostate cancer (PCa) ,52, colo2 at 37 °C.The following human cancer cell lines were used: MCF7 ; A549 ; PC9 ; SW480 ; SW620 ; CACO2 ; DU145 ; PC3 ; and LNCaP . All cell lines were obtained from ATCC . The corresponding media for each cell line was supplemented with 10% FBS , and additionally supplemented with 2 mM L-glutamine , 1% NEAA  in the case of DU145. Cells were grown in a humidified atmosphere containing 5% COαi signaling blocker pertussis toxin PTX, were evaluated in cellular proliferation and migration assays. Additionally, their ability to induce changes in the expression of certain proteins was assayed via qPCR. All antagonists and agonists were obtained from Tocris , and were dissolved in H2O according to the manufacturer’s instructions. When antagonists’ effects were evaluated, these effects were co-applied with nicotine or alone for the control experiments.Nicotine and three nAChR antagonists , together with the adenylyl cyclase inhibitor SQ22536 and the G® E-Plates . This system monitors changes in electrical impedance and records them as an increasing number of cells adhering to the surface of the plates. Depending on the cell type, 2 × 104 or 4 × 104 cells were plated in their corresponding complete growth medium with or without an agonist/antagonist, and proliferation was measured for 48–72 h in 15 min intervals. Statistical significance was analyzed using the Kruskal–Wallis test for non-parametric data with the GraphPad Prism (Version 9.3.1) software.Cell proliferation was evaluated as previously published by our group ,59 usingThe expression of diverse genes was evaluated following previously published procedures from our group . In brie4 cells were seeded in 0% serum media in the top chamber of xCELLigence® CIM-Plates . The respective culture media with 10% serum was added to the lowest chambers and served as a chemoattractant. For MCF7 cells, 1% serum was used in the top chamber. When compounds were being tested, these solutions were added to the top chamber. Afterward, migration was measured in 15 min intervals for 25 h. Statistical significance was analyzed using the Kruskal–Wallis test for non-parametric data with GraphPad Prism (Version 9.3.1) software.Transwell migration of cells was evaluated as previously published by our group  using thCHRNA5_7, target sequence CTGGTATCCGTATGTCACTTA; Hs_CHRNA5_6, target sequence CTGGACTCCACCGGCAAACTA; Hs_CHRNA5_5, target sequence ATGGATCACAGGTTGATATAA; and Hs_CHRNA5_4, target sequence CTGAGTAACAGCTAATCTTTA. Control cells were transfected with non-silencing siRNA (ns-RNA) , which presented no homology with any known mammalian gene. We performed the siRNA transfections using Interferin siRNA Transfection Reagent . Cells were incubated overnight with a transfection mix consisting of a combination of the corresponding serum-free media, the siRNA of interest or ns-RNA, and the Interferin solution. Afterward, the cells were exposed to treatments as specified in the drug treatment section, and the expression of diverse proteins was analyzed via qPCR. Treated cells were also used for proliferation and migration assays. Knockdown verification was performed 24 h or 48 h after siRNA transfection, and α5 expression was analyzed with qPCR.We performed the siRNA transfections with a mix of 4 different human α5 siRNAs using a 1:1:1:1 ratio in a final concentration of 0.12 nmol siRNA. All siRNAs were obtained from Qiagen. The α5 siRNAs used were as follows: Hs_6 cells suspended in nucleofection SE solution were transfected with the appropriate constructs using the DU145-specific program of the nucleofector. Next, cells were seeded and incubated for an additional 24 h before starting proliferation and migration experiments. Transfection verification was performed 24 h after nucleofection, and α5 expression was analyzed via qPCR and IF.The α5.pcDNA3.1/V5 and α5D398N.pcDNA3.1/V5 epitope-tagged constructs used for transfection were a generous gift from Dr. Larry S. Barak and have been described elsewhere . DU145 cTo positively ascertain the transfection of α5.pcDNA3.1/V5 and α5D398N.pcDNA3.1/V5 epitope-tagged constructs (a generous gift from Dr. Larry S. Barak)  into DU1First, to assess the expression of diverse nAChR subunits in various cancers, we analyzed data from the Human Protein Atlas , which iNext, we investigated the expression of α5 and other nAChR subunits that are known to mediate nicotine’s pro-oncogenic effects in different human cancer cell lines. Despite the fact that the maximum expression of the α5 was in urothelial and testicular cancer, our study focused on the cancer types with the highest incidence and mortality , namely Thus, the nAChR mRNA expression profiles demonstrate that α5 is the most significantly expressed subunit in lung, breast, CRC, and PCa cancer cells, albeit at different levels. Previous reports showed that α5 is upregulated in human lung cancer tissue , hepatocWe next studied whether nicotine alters proliferation in several types of human cancer cell lines through the activation of nAChRs. We tested nicotine-induced proliferation in the breast cancer cell line MCF7, the CRC line SW480, and the PCa cell line DU145. For this analysis, cells were incubated with increasing concentrations of nicotine, from 0.1 μM to 10 μM, for 72 h. These concentrations were previously used to study nicotine as an inducer of proliferation in cancer cells ,65. To dTo challenge the hypothesis that the increase in proliferation with 1 µM nicotine is caused by nAChR activation, proliferation was tested with nicotine in the presence of the non-specific nAChR antagonist mecamylamine (MEC). Since earlier work showed the relevance of α7 in nicotine’s pro-oncogenic effects , and becAdditionally, we evaluated whether nicotine, through nAChRs, affected migration in the DU145 cell line. Migration was investigated using an impedance-based assay with FBS as a chemoattractant . As showWhen migration was studied in the SW480 cells , a more CHRNA5 caused a ~90% decrease in transcripts in MCF7 and SW480 cells and a ~70% decrease in the DU145 cell line when evaluated at 24 h  ,70 and tNext, we studied how the silencing of α5 affected the nicotine-induced mRNA expression of PDL1, CD47, and both EMT markers in these cell lines. Cells were transfected with α5-siRNA or ns-RNA, and after 24 h, cells were incubated, or not, with 10 μM nicotine for 48 h before analyses. The results above clearly demonstrate that α5-containing nicotinic receptors are essential for nicotine-induced effects. As previous reports showed that the D398N mutation impairs α5′s function ,71, we nNext, proliferation in the absence or presence of 1 μM nicotine was studied in α5-WT- or α5-MUT-transfected DU145 cells. In α5-WT-transfected cells, 1 µM nicotine increased proliferation compared to non-treated cells C. In α5-In lung basal epithelial cells expressing the D398N mutation in α5, basal proliferation is increased through a mechanism involving adenylyl cyclase . Thus, wNext, we evaluated whether SQ or PTX affected nicotine-induced proliferation in non-transfected DU145 cells F. As shoUltimately, α5 is expressed in many different types of tissues. However, its physiological and pathophysiological roles are not fully understood. Recent findings have associated this subunit with attention , food reOur study also shows that nicotine can induce changes in the mRNA expression of EMT markers and immune regulatory proteins in all cell lines tested, specifically through α5. EMT is a vital step for the facilitation of a malignant and invasive phenotype in cancer, a process characterized by a decrease in epithelial proteins such as E-cadherin, as well as an increase in mesenchymal molecules such as fibronectin and vimentin . Nicotin2+ response to various nAChR agonists [αo and Gβγ dimers [2+ release from intracellular stores [2+ signals are, in part, mediated by a G-protein-mediated mechanism, ultimately leading to the release of Ca2+ from intracellular stores [2+ permeability or membrane depolarization in our present model.The non-synonymous single nucleotide polymorphism D398N of the α5 subunit, α5SNP, identified as a candidate mutation for smoking and lung cancer ,38, was agonists . Moreoveagonists . Whilst agonists  and are agonists ,89,90. Iγ dimers . Interesr stores  and may r stores . In thisr stores ,92. Furtr stores . This rer stores ,95,96,97Whether the endogenous α5 subunit is expressed on intracellular compartments in addition to the plasma membrane in the cell lines studied has not been addressed. In addition, despite the fact that one report has shown its expression in mitochondria , there aOur results point to α5-containing receptors as key signaling nodes of nicotine’s pro-oncogenic effects in diverse cancer types. This discovery is novel since, to date, no other reports have fully determined the importance of α5 in mediating nicotine’s oncogenic functions at the cellular level in breast, colon, or prostate cancer cells. Our findings show the need for α5 inhibitors/modulators to further investigate α5′s role in mediating nicotine’s pro-oncogenic effects in cancer and as a putative drug target. Yet further studies will have to be carried out in order to determine whether the expression of the α5SNP can be used in the future as a tumor-agnostic marker.Based on our findings, we show here that the α5 subunit is the key mediator of nicotine’s pro-oncogenic effects, whereas the silencing of the subunit by siRNA rendered the cells studied irresponsive to this stimulus. The signaling events mediated by this subunit are carried through a G- protein mediated mechanism. Most remarkably, the introduction of the α5SNP rendered the cells (i) irresponsive to nicotine, an effect reminiscent of the one observed when the mutation is present in neurons or heterologously expressed receptors in HEK cells ,40,41,42


Francisella tularensis (Ft), a pathogenic intracellular gram-negative bacterium that infects a wide range of animals and causes severe disease and death in people, making it a public health concern. Vaccines are the most effective way to prevent tularemia. However, there are no Food and Drug Administration (FDA)-approved Ft vaccines thus far due to safety concerns. Herein, three membrane proteins of Ft, Tul4, OmpA, and FopA, and a molecular chaperone, DnaK, were identified as potential protective antigens using a multifactor protective antigen platform. Moreover, the recombinant DnaK, FopA, and Tul4 protein vaccines elicited a high level of IgG antibodies but did not protect against challenge. In contrast, protective immunity was elicited by a replication-defective human type 5 adenovirus (Ad5) encoding the Tul4, OmpA, FopA, and DnaK proteins  after a single immunization, and all Ad5-based vaccines stimulated a Th1-biased immune response. Moreover, intramuscular and intranasal vaccination with Ad5-Tul4 using the prime-boost strategy effectively eliminated Ft lung, spleen and liver colonization and provided nearly 80% protection against intranasal challenge with the Ft live vaccine strain (LVS). Only intramuscular, not intranasal vaccination, with Ad5-Tul4 protected mice from intraperitoneal challenge. This study provides a comprehensive comparison of protective immunity against Ft provided by subunit or adenovirus-vectored vaccines and suggests that mucosal vaccination with Ad5-Tul4 may yield desirable protective efficacy against mucosal infection, while intramuscular vaccination offers greater overall protection against intraperitoneal tularemia.Tularemia is a highly contagious disease caused by infection with  Francisella tularensis (Ft), a nonmotile, intracellular gram-negative coccobacillus, is the causative agent of fatal tularemia disease, which is endemic throughout most of Europe, northern and central Asia and North America , Ft subsp. holarctica (type B), Ft subsp. mediasiatica and Ft subsp. Novicida   , outer mde (LPS)  and Tul4de (LPS) ) and intFt-induced disease, respiratory tularemia.In the present study, we performed a comparative evaluation of protective immunity against tularemia provided by subunit or Ad5-vectored vaccines, and suggested that mucosal vaccination using Ad5-Tul4 may provide desirable protective efficacy against the most severe Francisella with a normal genome size and number of protein-encoding sequences (CDS) was obtained from the NCBI database. The mean genome size was 1.80 Mb (1.78-1.9 Mb), and the average number of CDS was 1,519 , “replication, recombination and repair”  and “amino acid transport and metabolism”  were the top three largest functional groups  immunization showed no superiority  and to a greater extent fragment 18 (RLQWQAPEGSKCHDT) by producing IFN-γ, demonstrating that the Tul4 antigen was indeed processed and presented to T cells, eliciting a potent cellular immune response  was observed when mice were immunized with Ad5-OmpA, Ad5-FopA, or Ad5-DnaK  3 weeks after the second immunization. Five days post-infection, the spleen, liver and lung were harvested, and the expression of Ft LVS-specific AKR DNA was assessed using real-time PCR as a measurement of bacterial burden in the tissues. Compared to the i.m. immunized mice, i.n.-immunized mice showed a significantly lower bacterial dissemination in the lung and spleen upon i.n. challenge, with a 73.86% Ft LVS load reduction in the lung, 92.81% in the spleen and 95.16% in the liver    . Humaniz6 CFU) . The faiesponses . Furtheresponses . The potnfection .Ft infection is the intranasal route, which results in pneumonic tularemia with a mortality rate of up to 60% without therapeutic intervention. Bacteria dissociate from the respiratory tract, and continued replication in other organs results in septicemia and cytokine storms, which lead to overwhelming tissue damage and eventual death . The bacteria were diluted to achieve a final concentration of 2× 104 CFU/ml or 4× 104 CFU/ml, and these suspensions were used in all challenge experiments.The g, China , handledE. coli BL21 competent cells, and expression was induced by the addition of IPTG  when the OD600 reached 0.8-1.0. The identity of the purified rTul4, rFopA, and rDnaK proteins was examined by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) with Coomassie Blue staining. The concentration of each recombinant protein was estimated by a BCA Protein Assay Kit  according to the manufacturer’s instructions.pTIG-Trx vectors were constructed with the codon-optimized Tul4, FopA, and DnaK genes. Then, the vectors were transferred to Recombinant replication-defective human type 5 adenoviruses were constructed as described previously . Briefly3  and 25 μg of CpG1826  adjuvants. For Ad5-based vaccine administration, mice were immunized i.m. (100 μl) or i.n.  on day 0 or on days 0 and 21 with 1x108 plaque-forming units (PFU) of the indicated adenoviruses. For challenges, intraperitoneal (i.p.) injection  or intranasal inhalation  of Ft LVS were applied at the indicated time.Animal experiments were performed according to the guidelines of the Institutional Experimental Animal Welfare and Ethics Committee. Female BALB/c mice aged 8-10 weeks  were used for all experimental groups. For subunit vaccine administration, mice were immunized via the s.c. route on days 0, 14 and 28 with 10 μg of the indicated proteins together with 100 μg of Al(OH)Ft burden was evaluated with real-time PCR as previously described . Five microliters of purified DNA was amplified in a 20 μl reaction containing 10 μl of 2×TaqMan Universal Master Mix II, 0.8 μM forward primer, 0.8 μM reverse primer, and 200 nM probe. A forward primer (5’-GCAGGGCGAGCACCATT-3′), reverse primer (5′-ATCTTGCATGGTCACCACTTGA-3’), and probe (5’-FAM-CGATATTTGCCTGTTAGCACTCCT-Tamra-3’) were used. Reactions were incubated at 50°C for 2 min, followed by 95°C for 10 min, and then thermal cycled for 40 cycles (95°C for 15 s and 60°C for 1 min).escribed . BrieflyFt (iFt) at 4°C overnight. After successive antibody incubation with 1:20000-diluted HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2a  and washing with PBST, the assay was then developed for 6 min with 100 μl of TMB substrate solution , stopped by the addition of 50 μl of stop solution , and measured at 450 nm/630 nm . The endpoint titer was defined as the highest reciprocal serum dilution that yielded an absorbance > 2-fold over the optical absorbance value of the negative control.For measurement of IgG (IgG1 and IgG2a) and IgM titers, serum from immunized mice was serially diluted and added to 96-well microplates , which were precoated with 2 μg/ml indicated antigen proteins or 4% paraformaldehyde-inactivated 5 splenocytes were seeded in 96-well plates and treated with purified Tul4 protein for 72 h at 37°C with 5% CO2. Then the supernatant was collected and mixed with Capture Beads and the indicated cytokines were then quantified by flow cytometry.A cytometric bead array (CBA) mouse Th1/Th2/Th17 cytokine kit (BD Biosciences) was used for cytokines detection according to the manufacturer’s protocols. Briefly, a total of 2×105 splenocytes from immunized mice were stimulated with 31 synthesized peptides  and seeded in precoated ELISpot plates for 48 h at 37°C with 5% CO2. After successive antibody incubation and washing, the plates were measured on the AT-Spot 2100 reader .A mouse IFN-γ enzyme-linked immunosorbent spot (ELISpot) kit  was used for T-cell epitope identification according to the manufacturer’s protocols. Briefly, a total of 2×10All of the statistical analyses were performed using GraphPad Prism 8.0.2 software. Comparisons among the groups were analyzed by one-way analysis of variance (ANOVA) unless otherwise specified. For survival analysis, the log-rank (Mantel−Cox) test was used. Antibody titer data were log transformed before analysis. Data are shown as the geometric mean with geometric SD. n.s., not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Care and Use Committee of the Beijing Institute of Biotechnology.JX, RY and WC designed, directed and supervised the entire study. MZ performed most of the experiments and processed data. YZ analyzed the results and wrote the manuscript. XZ performed the immunoinformatic analysis. YM, EH, ZW, YLi, KL, and YLiu assisted in the immunization and evaluation experiments. All authors contributed to the article and approved the submitted version.


Aesculus chinensis) is an important medicinal tree that contains various bioactive compounds, such as aescin, barrigenol-type triterpenoid saponins (BAT), and aesculin, a glycosylated coumarin. Herein, we report a 470.02 Mb genome assembly and characterize an Aesculus-specific whole-genome duplication event, which leads to the formation and duplication of two triterpenoid biosynthesis-related gene clusters (BGCs). We also show that AcOCS6, AcCYP716A278, AcCYP716A275, and AcCSL1 genes within these two BGCs along with a seed-specific expressed AcBAHD6 are responsible for the formation of aescin. Furthermore, we identify seven Aesculus-originated coumarin glycoside biosynthetic genes and achieve the de novo synthesis of aesculin in E. coli. Collinearity analysis shows that the collinear BGC segments can be traced back to early-diverging angiosperms, and the essential gene-encoding enzymes necessary for BAT biosynthesis are recruited before the splitting of Aesculus, Acer, and Xanthoceras. These findings provide insight on the evolution of gene clusters associated with medicinal tree metabolites.Horse chestnut ( Aesculus chinensis) is a tree species that can produce medicinal compounds such as aescin and aesculin. Here, the authors assemble its genome, identify key genes involved in the biosynthesis of these two group of compounds, and achieve the de novo synthesis of aesculin in E. coli.Horse chestnut ( With the growing body of genome sequence information available, multiple investigations have been completed to clarify the collinear BGC organization within and across species9. Examples include triterpene thalianols in Arabidopsis thaliana and its close relatives, benzylisoquinoline alkaloids in Papaver, steroidal glycoalkaloids in Solanaceae, triterpene cucurbitacins in Cucurbitaceae, and momilactone diterpenoids in grass13. Evidence from herbaceous plants showed that complex genetic events, such as whole genome duplication (WGD), tandem duplication, gene relocation, chromosomal inversion, and lateral gene transfer, impact the assembly, maintenance, and diversification of genes that are physically clustered and nonhomologous but functionally coordinated during the evolutionary process17. In contrast, examples that demonstrate genomic mechanisms leading to the exclusive production of pharmaceuticals in trees are limited and include soapbark, yew, and happy tree20.Plants synthesize a wide variety of structurally diverse medicinal compounds, such as noscapine, casbenes, avenacins, cucurbitacins, and taxadiene, which are exclusive to specific lineages or species. Interestingly, the biosynthesis of numerous compounds results from a biosynthesis-related gene cluster (BGC), which is characterized by physically colocalized genes involved in a common metabolite pathwayAesculus L. in the soapberry family (Sapindaceae) is native to the temperate northern hemisphere21. This plant accumulates medicinal triterpenoid saponins  and the coumarin compound aesculin, all of which possess medicinal properties25. Escin Ia belongs to barrigenol-type (BAT) triterpenoid saponin, which is derived from an oleanane (β-amyrin) backbone; this backbone further undergoes region-specific C-16, C-21, C-22, C-24, and C-28 oxygenation to become protoaescigenin, the main aglycone of aescin. Protoaescigenin is further modified by the addition of a glucuronyl moiety at the C-3 position, as well as diglucoside and 2-methylcrotonylation at the C-21 position and acetylation at the C-22 position to form escin Ia27. Aescin preparations have been administered orally, intravenously (sodium aescinate containing escin Ia and escin Ib), and topically in clinical trials to treat chronic venous insufficiency, edema, and hemorrhoids29. Aesculin, which is 6,7-dihydroxycoumarin 6-O-glucoside, along with digitoxin, has been used as a popular eye drop solution to alleviate symptoms, such as asthenopia, eye pain, and diplopia33. Currently, the biosynthesis of aescin and aesculin in horse chestnut trees, as well as the emergence of the BAT biosynthetic pathway in this lineage, remains uninvestigated.A. chinensis. We reveal an ancient whole-genome duplication that occurred ~30.8 million years ago (MYA) in Aesculus, which might have contributed to the formation of BAT triterpenoid-related BGCs. Furthermore, we conduct functional verification of the key enzymes involved in the biosynthesis of escin Ia, such as AcOCS6, AcCYP716A278, AcCYP716A275, AcCSL1, and AcBAHD6, as well as the enzymes crucial for aesculin biosynthesis, namely, Ac4CL1-3, AcF6’H1-2, AcUGT84A56, and AcUGT92G7. In addition, we utilize a comparative genomics-driven approach to clarify the organization and evolution of BAT BCGs across angiosperms.In this work, to determine the biosynthesis pathway of these compounds, we assembled a chromosome-scale genome of 1), escin Ia (2), escin Ib (3), esculetin (4), and aesculin (5) in branches, flowers, leaves, pericarps, and seeds , and the contents of 2 and 3 were the highest in seeds (p < 0.05)  massively accumulated in the cotyledons near the testas    and Dimocarpus longan (52.87%). Of these repetitive elements, 24.37%  of TEs were long terminal repeat (LTR) retrotransposons, of which 98.1% belonged to the Gypsy superfamily (32.7%) and Copia superfamily (65.4%)  of repetitive DNAs in the ongan 52.7%. Of thA. chinensis is closely related to other sequenced Sapindaceae species, X. sorbifolium and D. longan  from the range of 21.8 MYA to 50.4 MYA. The split between A. chinensis and X. sorbifolium occurred at ~32.5 MYA with a 95% CI from 17.5 MYA to 45.4 MYA  for paralogous genes and anchor pairs in collinear regions of A. chinensis showed a clear peak at approximately 0.24 and a minor peak at ~1.75, suggesting that the A. chinensis genome might have experienced two WGD events  event. Our analysis confirmed that only one KS peak was detected in C. clementina and X. sorbifolium, at approximately 1.5 and 1.75, respectively. This ancestral KS peak, shared by the A. chinensis, C. clementina, and X. sorbifolium genomes, represents the γ-WGT event. The KS distribution of orthologs between A. chinensis and X. sorbifolium showed one KS peak at ~0.25, slightly larger than the KS value of paralogs in A. chinensis. This result again suggests that the recent WGD event in A. chinensis occurred after the divergence between A. chinensis and X. sorbifolium. Using the divergence time and mean KS value of orthologs between A. chinensis and X. sorbifolium, between A. chinensis and C. clementina, and between A. chinensis and V. vinifera, we estimated that the species-specific WGD event in A. chinensis (Aα) occurred at ~30.8 ± 1.33 MYA  and the regulation of these responses  event in the ome Fig. . Collineina Fig. . These rMYA Fig. . Further37. WGD is the major evolutionary force for the production of phenotypic diversity, speciation, and domestication38. Examples of WGD-derived plant phenotypic and metabolic diversity that contributes to species-specific gene expansion include oil biosynthesis in wild olive tree domestication39, ursane triterpene synthesis in loquat (Eriobotrya japonica) domestication40, camptothecin production in happy tree (Camptotheca acuminata) domestication19, and triptolide content in thunder god vine (Tripterygium wilfordii)41. To investigate the impact of the A. chinensis-specific WGD event on aescin biosynthesis, we systematically calculated Ks for each duplicated paralogous gene pair, emphasizing the upstream pathway genes involved in terpene biosynthesis   are triterpene saponins, biosynthesized from the 30-carbon intermediate 2,3-oxidosqualene (6) by the sequential actions of multiple enzymes, including OSC, P450s, UGTs, and acyltransferases (Supplementary Table 43. The pentacyclic triterpene skeleton is derived from a dammarenyl cation and D and E ring expansion en route to β-amyrin (7). The 7 further undergoes site-specific oxidation catalyzed by P450s, forming diverse nonglycosylated aglycones, which are collectively referred to as 122. Acylation and glycosylation of 1 contribute to the structural diversification of triterpene saponins  known to act in such metabolism. As a result, four BGCs containing OSCs and one BGC containing CYP716 genes were discovered, which are known as AcClusters I–V; these BGCs were then implicated in triterpenoid biosynthesis in the A. chinensis genome , seven P450s , and five BAHDs (AcBAHD1-AcBAHD5) within a 350-kb region. AcCluster II is located on chromosome 8 and consists of two P450 genes (AcCYP716A275 and AcCYP716BX2) and one cellulose synthase-like gene (AcCSL1) (Supplementary Data AcOSC6), four P450s , and three BAHD genes  were abundantly expressed in A. chinensis seeds, in agreement with the high accumulation of 2 and 3 in the seeds, which putatively participate in aescin formation via the triterpene cyclization, hydroxylation, glucuronidation, and acylation  in Nicotiana benthamiana, which does not naturally generate 7, led to the production of 7 in plants as measured by GC–MS/MS and confirmed by comparing mass spectral fragmentation with an authentic standard , as similarly verified by comparison to the known production by AcOSC6/BfCYP716Y144 , which is known to be produced by AstHMGR/AcOSC6/GmCYP72A6945 families. Identical compounds produced by distant species may originate from different enzymes; however, these compounds more often arise via the same pathway, regardless of whether these enzymes are homologous61.Heterologous overexpression of full-length ard Fig. . The funard Fig. . To furt144 Fig. . Accordi945 Fig. . AcCYP7164. Recent reports have identified a series of cellulose synthase-derived glycosyltransferases (CSyGT) that transfer the glucuronic acid moiety to the C-3 position of triterpenoid aglycones in the following leguminous plants: G. max, Glycyrrhiza uralensis, Lotus japonicus, and Spinacia oleracea66. We constructed the phylogenetic relationship of AcCSL1 with characterized and related proteins from different plants available in the literature65. AcCSL1 is clustered with a clade of characterized proteins in a CslM subfamily involved in glucuronidation at the C-3 position of oleanane-type triterpenoids, suggesting a similar function in A. chinensis  in AcCluster I and AcBAHD6 in WGCNA towards substrate 1 with acetyl-CoA as the donor, in vitro enzyme assays were performed using recombinant proteins from E. coli  through NMR analyses , resulting in the formation of 2 (C22-O-acetylation of 12). This result was confirmed by comparing the retention time and MS/MS spectra of the product and an authentic standard , β-BA, and 11-keto-β-BA, thus forming all the major C3a-O-acetyl-BAs 70. Phylogenetic reconstruction further confirmed that the BsAT1 is the closest ortholog of AcBAHD1, 3, 5, and 6, which are placed with the clade IIIa BAHDs representing ATs that act on distinct acceptors , a coumarin , is produced via the phenylpropanoid pathway and distributed in Aesculus, Fraxinus, and Populus71. The relevant upstream genes identified in this study from the A. chinensis genome include seven phenylalanine ammonia-lyases (PALs), three cinnamic acid 4-hydroxylases (C4Hs), five cinnamic acid 3-hydroxylases (C3Hs), four 4CLs, and five F6’Hs  and four AcF6’H (AcF6’H1-4) were cloned based on their expression patterns and functionally characterized. Our findings revealed that Ac4CL1 exhibited a broad expression pattern, whereas Ac4CL2 and Ac4CL3 were narrowly expressed in the pericarp. The level of AcF6’H1 expression was high in the pericarp and branch, but low in the flower, while AcF6’H2-4 was narrowly expressed in the flower. Enzyme assays using spectrophotometry demonstrated that all three characterized Ac4CL enzymes had a catalytic affinity for caffeic acid (13) to be caffeoyl-CoA (14)  Fig. . Notablye 4 Fig. . CompareE. coli to produce recombinant proteins; the enzymatic activities of these proteins were then investigated using 4 as acceptors and UDP-glucose as a donor substrate  was stronger than that of AcUGT84A56 (KM = 177.25 μM) for 4  and erythrose 4-phosphate can be increased by over-expressing ppsA and tktA, respectively72; in addition, tyrosine production can be improved by over-expressing feedback inhibition-resistant variants of tyrA and aroG73. In this study, four key enzymes were expressed to enhance the carbon flux in the shikimate pathway.The characterized enzymes Ac4CL2, AcF6’H1, and AcUGT92G7 were used to assemble a de novo biosynthetic pathway for compound 5, we chose BWΔpykAΔpykF as the basal strain to reduce the conversion of PEP to pyruvate74. BWΔpykAΔpykF was co-transformed with plasmids pZE-649T and pCS-TPTA-HpaBC, forming strain BW1. M9 medium supplemented with 5 g/L yeast extract was employed for de novo production of 5. The corresponding fermentation curves and titer of 5 are shown in Fig. 600 of 18.1 at 72 h. The production of 5 also increased steadily as fermentation proceeded. The final production of 5 at 72 h reached 16.3 ± 0.7 mg/L. Therefore, we reconstructed a biosynthetic pathway for 5 by combining three genes from A. chinensis. Additionally, these pathway engineering strategies could provide powerful tools and insights to further progress and discovery in coumarin biosynthesis.For de novo synthesis of 75. The current genome available is abundant and can provide crucial clues into how genes that encode enzymes from a common biosynthetic pathway are assembled into a formation of BGCs9. Comparative genomics in combination with functional characterization indicates that early-arising BGCs underwent dynamic changes in auxiliary enzymes to generate structurally diverse triterpenoids within and between Arabidopsis or between cucurbits75. However, little is known about the evolutionary trajectories of triterpenoid BGCs in other plant clades.The origin of plant BGCs stems from the assembly of genes after gene duplication, neofunctionalization, and genomic relocationAcOSC6, CYP716, BAHD IIIa, and CSL genes distributed in AcCluster I and AcCluster II are crucial enzymes for the catalysis of BAT biosynthesis. The mean KS value of 17 paralogous gene pairs localized in the syntenic blocks of AcCluster I and AcCluster II was 0.27, which is more generally similar to the KS value of the paralogs in A. chinensis (0.24), suggesting that the duplication event of AcCluster I and AcCluster II might originate from the A. chinensis-specific WGD event, Aα WGD. Syntenic analysis showed that AcCluster I and AcCluster II are conserved among Hippocastanoideae species, including A. chinensis, Acer yangbiense, and X. sorbifolium , but these lineages only contain one CAS gene, not the BAT biosynthesis-related BAS, CYP716, CSL, and BAHD genes. The CYP716A member first appeared in the corresponding region of species within Ranunculales, i.e., Papaver somniferum. However, the syntenic region is not present in all examined Superasterid species. After the split with Superasterids species, almost all the syntenic segments retain at least one OSC gene except for Cruciferae representatives, such as A. thaliana and B. rapa in Superrosids. Remarkably, a sizeable species-specific tandem duplication of 10 complete and 10 partial OSC genes was found in the conserved syntenic region of the V. vinifera genome. Complex insertion, deletion, duplication, and arrangements of OSC, CYP716, BAHD, and CSL genes in the syntenic regions of Superroside species were observed. The BAS/CYP716/BAHD BGC was always found in the syntenic region of Superrosid species, but dynamic duplication and reorganization occurred . In addition, the BGC underwent further dynamic evolution during speciation, such as the tandem gene duplication of CYP716A and CYP716BX and the Aesculus-specific duplication of AcCluster I and AcCluster II from the Aα WGD event observed here.Further large-scale analysis of evolutionary dynamics for this BAT BGC among 21 published plant genomes, including early-diverging angiosperm, monocot, and eudicot genomes, was conducted to examine how this syntenic region may have evolved Fig. . An ~1-Mred Fig. . We propOSCs, P450s, CSLs, and BAHDs, we generated a series of phylogenetic trees using the maximum-likelihood method; this procedure was based on multiple alignments of protein sequences from the aforementioned species for synteny analysis . These results indicated that CSL genes localized in the BAT BCGs of A. chinensis, A. yangbiense, and X. sorbifolium were recruited to catalyze glucuronidation activity before their speciation.To further examine the evolutionary origins of sis Fig. . The phyCAS and one BAHD IIIb in A. trichopoda; (2) Death in monocots: The region containing CAS and other non-BAT functional genes is retained, but BAHD IIIb is lost; (3) Evolution in specific early-diverging eudicots: BAS undergoes evolution and CYP716 is added; (4) Death in Superasterids: The BGC is lost in this lineage; (5) Assembly of the BAT BGC in Hippocastanoideae: This functional BGC (BAS/CYP716/CSL/BAHD) is formed, leading to BAT production; (6) Reorganization and diversification of the BAT BGC: This BGC undergoes dynamic evolution in a species-specific manner via tandem duplication and WGD , the Chinese Academy of Medical Sciences (CAMS), China. N. benthamiana was cultivated for a duration of ~4 weeks under an 18 h light/6 h dark cycle within a greenhouse located at the National TCM Gene Bank, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. High-quality standards for protoaescigenin (1), desacetylaescin I (12), escin Ia (2), escin Ib (3), esculetin (4), and aesculin (5) were purchased from Chenguang Biotechnology Inc. . Caffeic acid (13), disodium 2-oxoglutarate, caffeoyl-CoA (14) were purchased from Chengdu Push Bio-technology Co., Ltd . UDP-Glucose (UDPG), UDP-Glucuronic acid (UDPGA), adenosine 5′-triphosphate, and S-adenosylmethionine (SAM), acetyl-CoA were purchased from Sigma-Aldrich (USA). All chemicals used were of analytical or HPLC grade.A. chinensis capsules were harvested and promptly encased in a 10% gelatin (w/v) solution. Initially, the tissues were positioned within Tissue-Tek cryomolds (25 × 20 × 5 mm) and enveloped by the gelatin solution. Then, the molds were transferred to a −80 °C freezer for 1 h to solidify into blocks. During cryo-sectioning, the sample blocks were affixed directly onto the sample holder of a cryostat  using deionized water as an adhesive. Sections with a thickness of 16 µm were obtained at −20 °C and immediately affixed onto indium tin oxide-coated glass slides for subsequent imaging analyses. To preclude condensation, the tissue sections underwent a vacuum desiccation process for ~10 min before the matrix was applied. Optical images of the sections were captured utilizing a Zeiss Axio M2 microscope . For consistent matrix application, a custom-designed automated pneumatic-assisted system was employed. The matrix application system and coating procedure were conducted86. In brief, a solution of 50 mg/mL 2,5-dihydroxybenzoic acid dissolved in acetonitrile:H2O   was applied for positive mode MALDI experiments. The MALDI system  was operated under atmospheric pressure and was coupled with a Q Exactive HF Orbitrap mass spectrometer . To ensure uniform deposition onto the capsule samples, the nebulizer was situated 3 cm above the sample and oscillated over the Plate 100 times. The flow rate was set at 6–8 mL/h, while the gas pressure was adjusted to 50 psi to facilitate the delivery and nebulization of the matrix solution.Fresh m/z range of 158.9640–1926.6365. Instrument calibration was performed using the ESI portion of the instrument with NaTFA clusters and a quadratic calibration of the clusters. Data analysis was executed utilizing the SMALDIControl software package .Each single-scan spectrum was composed of 100 accumulations of laser pulses at a frequency of 1 kHz, employing a “small” laser focus configuration. MALDI images were acquired with a spatial resolution of 50 µm. The instruments were calibrated utilizing the ESI inlet, employing sodium trifluoroacetate (NaTFA) clusters spanning the 2O mixture  was introduced into 50 mL centrifuge tubes, followed by a 30 min sonication period utilizing an 8891 ultrasonic cleaner . Subsequently, the extracts were centrifugated at 14,000 × g for 5 min, and the supernatants were collected. These extract solutions were employed for subsequent analyses. For the quantification of 1,2,3,4, and 5, LC–QQQ–MS/MS assays were executed utilizing a 6470 Triple Quadrupole mass spectrometer  coupled with an Advance 1290 UHPLC system . Analytes were resolved using an Agilent Eclipse Plus C18 column . Mobile phase A comprised 0.1% acetic acid, while phase B was composed of H2O containing 0.1% acetic acid. The gradient elution program was as follows: 0–25 min, A:B = 68:32. A 1 μL volume was injected, and the flow rate was set at 0.30 mL/min, with the column temperature maintained at 30 °C. The mass spectrometer, which was operated in either positive or negative ion mode, was configured for multiple reaction monitoring (MRM) mode. For each compound, two precursor-product ion MRM transitions were selectively monitored. Data acquisition and subsequent analysis were executed employing MassHunter software , facilitating the quantification of all seven metabolites for which corresponding standards were available.All tissues were subjected to vacuum freeze-drying, and each individual sample was precisely weighed to 100 mg and subsequently pooled for subsequent solvent extraction. To effectuate this process, 10 mL of a methanol:HA. chinensis using Solanum lycopersicum (900 Mb) and Brassica rapa (485 Mb) as the standards on a BD AccuriTM C6 . In addition, the genome size of A. chinensis was also estimated according to k-mer frequency (k-mer length 17) of genome sequencing data using Jellyfish (v.2.0).Flow cytometry was employed to estimate the genome size of A. chinensis leaves was executed through a plant DNA extraction kit . Short-read paired-end libraries were constructed following the manufacturer’s guidelines and sequenced on an Illumina Hiseq X-ten platform . For long-read sequencing, high-quality A. chinensis genomic DNA was employed for the preparation of 20 kb insert libraries, adhering to standard ONT library preparation protocols. These libraries were then sequenced utilizing the ONT GridION X5 platform . Base-calling of the raw ONT reads was accomplished using the Oxford Nanopore base caller Guppy (v.1.8.5) with default parameters. The ONT reads were subjected to correction, trimming, and assembly into genomic contigs using CANU (v.1.5)87 and SMARTdenovo88. The assembled contigs were further polished three times utilizing Illumina short reads using Pilon . The completeness of the genome assembly was estimated using Benchmarking Universal Single-Copy Orthologs  with Embryophya odb10 dataset. A Hi-C library of young A. chinensis leaves was prepared by Annoroad Genomics (http://en.annoroad.com) following the standard procedure89. The Hi-C sequencing data was aligned to the assembled contigs by BWA-MEM (v.0.7.17), and then the contigs were clustered onto chromosomes with 3D-DNA.DNA isolation from A. chinensis RNA samples were meticulously extracted from diverse tissues, encompassing young leaves, branches, flowers, pericarps, and seeds, using the RNeasy Plant Mini Kit . These RNA samples were then employed to construct RNA-Seq libraries utilizing the TruSeq RNA Library Prep Kit v.2 . Subsequently, the libraries were subjected to sequencing using the NextSeq 500 platform. The quantification of transcript abundance was gauged by calculating the Fragments per Kilobase of the exon model per Million mapped reads (FPKM) values. This quantification was executed through the utilization of HISAT2 (v.2.0.5) alignment and FeatureCounts (v.1.6.3). Employing the FPKM values derived from different tissues, a weighted gene co-expression network analysis (WGCNA) was performed to infer networks of co-expressed genes. Module eigengene values were computed within the WGCNA framework and subsequently correlated with the accumulation profiles of potential metabolites across distinct plant tissues.For RNA-Seq analysis, A. chinensis genome were explored through de novo-based and homology-based strategies, facilitated by RepeatModeler (v.1.0.9) software. For de novo repeat identification and classification, the LTR_Finder and LTR_retriever programs were employed to identify and classify repeat elements. De novo transcript assembly was performed using Trinity (v. 2.2.0)90 with default parameters, and peptide sequences were predicted by TransDecoder . The MAKER annotation pipeline was used for ab initio predictions of protein-coding genes in the masked A. chinensis genome (v.2.31.9)91.Repetitive sequences within the A. chinensis. Protein sequences from characterized proteins in triterpenoids and coumarins were used to identify candidate genes by BLASTP  in the A. chinensis genome. The identified candidate genes were verified by HMMER; only proteins with representative domains were retained.BLASTP-based and HMMER annotations in combination were used to find high-confidence proteins associated with triterpenoid and coumarin biosynthesis in A. chinensis with M. domestica, C. sativus, G. max, P. trichocarpa, C. clementina, D. longan, X. sorbifolium, B. rapa, A. thaliana, T. cacao, G. raimondii, and V. vinifera was constructed using RaxML (v.8.2.10)92 using the JTT + G + I substitution model for amino acids with 1000 bootstrap replicates. The divergence time of selected species was calculated using the program MCMCTREE in PAML, according to fossil divergence time points for the specific Sapindaceae and Rutaceae (21.8–50.4 MYA) split obtained from TimeTree (http://www.timetree.org). Then, CAFE (v.2.1) was utilized to identify contraction and expansion analyses of these gene families following species divergence prediction under a probabilistic graphical model. MCScan (Python version) was applied to perform genome synteny analysis.A maximum-likelihood (ML) phylogenetic tree of single-copy genes, which were clustered into orthologous groups, from the genomes of http://plantismash.secondarymetabolites.org/) and a manual search to annotate the triterpenoid BGCs. To assign BGCs to triterpenoid biosynthesis, we surveyed whether these genes contained one or more OSC genes and annotated the 20 surrounding genes that were physically adherent. The obtained BGC containing OSC6 was mapped to the self-genome and four other genomes  to uncover collinear segments using MCScan (python version).We used the plantiSMASH online pipeline , 19 AcUGTs and AcF6’Hs , and three Ac4CLs  were amplified using KOD-Plus-Neo DNA polymerase , and the primers were listed (Supplementary Tables AtHMGR (KY284573), GmCYP72A69 (NM_001354946), BfCYP716Y1 (KC963423.1) and GmCSyGT1  were synthesized (GenScript Biotech).Total RNA was extracted using an EasyPure® Plant RNA Kit . First-strand cDNA was synthesized from total RNA using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix cDNA synthesis kit  according to the manufacturer’s protocol. Full-length ORFs of AcOSC6 and P450 genes were first recombined into the donor vector pDONR207 and then subcloned and inserted into pEAQ-HT-DEST1 using the LR clonase II enzyme . pEAQ-HT-DEST constructs were electrotransformed into the Agrobacterium tumefaciens (EHA105) strain. Transformed A. tumefaciens strains carrying the candidate genes were infiltrated solely or equally in combination with AtHMGR into N. benthamiana using a syringe. Infiltrated N. benthamiana leaves were collected for metabolite analysis after 4–5 days. The infiltrated N. benthamiana was lyophilized, ground, and weighed to 10 mg. The powder was extracted with 1 mL saponification solution (EtOH:H2O:KOH 9:1:1 v:v:w) at 65 °C for 2 h. The extracts were then evaporated until dry and re-extracted with 500 μL ethyl acetate by shaking for 2 min. After an equal volume of H2O was added, the samples were vortexed, and 100 μL of the upper organic phase was transferred to a fresh vial and dried. Trimethylsilylation was carried out with 50 μL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide . The trimethylsilylation samples were analyzed using GC‒MS.BTS1 and ERG27 genes using CRISPR/Cas9. Gene cassettes of Ppgk-tHMG-Tadh1, Ptef1-ERG1-Tpgk, Ptdh3-ERG20 + ERG9-TCYC1, and PpgK-β-AS-Tadh1 were amplified from the corresponding plasmids. Then, these cassettes and the URA3 marker gene were integrated into the delta site of strain Y0. The strain producing the highest 7 was designated strain Y1-20-6. The ORFs of AcCYP716A275, BfCYP716Y1, AcCYP716A278, and GmCYP72A69 were subcloned and inserted into the yeast expression vector pESC-HIS, and the recombination constructs were cotransformed into yeast strain Y1-20-6. The metabolite extract was followed as above for GC‒MS analysis.Yeast strain Y0 was derived from strain Cenpk2-1D, which was purchased from EUROSCARF, by knocking out AcCSL1 or an empty vector were transformed into yeast S. cerevisiae WAT11. The recombinant yeast strains were first grown in 20 mL SD-His medium containing 20 g/L glucose at 30 °C for 24 h. 200 μL of strain solution was used with urine glucose test strips to ensure that glucose was completely digested. After the glucose test, the yeast cells were harvested by centrifugation at 1000 × g for 5 min and washed twice with ddH2O. SD-His medium with 20 g/L galactose was used to resuspend the strains. One milliliter of each strain was used for the yeast in vivo reaction. The substrate 1 was supplemented at 100 μM into the cultures. After 16 h, ethyl acetate was used to stop the reaction, and the supernatant was concentrated at low pressure after ultrasonic centrifugation until the ethyl acetate was completely volatilized. The pellet was dissolved in 200 μL methanol and stored at 4 °C for further UPLC‒MS analysis. Microsomal proteins were isolated and dissolved in a protein storage buffer . The enzymatic activity of AcCSL1 was assayed in 100 μL of reaction volume, which contained 35 mM Tris–HCl (pH 7.4), 1 mM dithiothreitol, ~30 μg of microsomal proteins, 100 μM 1, and 1 mM UDPGA. The assays were incubated for 1 h at 30 °C. The reactions were stopped using 100 μL of methanol. Finally, the extracts were centrifuged for 10 min at 14,000 × g and analyzed by UPLC–MS.The yeast expression vector pESC-HIS constructs with UGT and BAHD genes from A. chinensis were obtained using KOD Plus-Neo according to the kit instructions . PCR products of UGT genes were purified with an EasyPure Quick Gel Extraction Kit , digested with the corresponding restriction enzyme, and ligated into similarly predigested pMAL-c2x vectors or pET28a . The recombinant MBP- or His-fusion proteins were purified using maltose resin according to the manufacturer’s instructions . To analyze the activity of nineteen recombinant UGTs in vitro, 100 µl final reactions were adopted consisting of 10 mM DTT, 50 mM Tris–HCl (pH 7.0), 0.1 mM substrates, 1 mM UDP-glucose as a sugar donor, and recombinant proteins (5–10 µg). After incubation for 1 h at 37 °C, the reactions were stopped with 100 μL methanol. Products were analyzed by HPLC after centrifugation at 14,000 × g for 10 min and filtration through a 0.22 μm micropore filter. The KM and Vmax of AcUGTs were measured using 1 mM UDP-Glc as the sugar donor and 4 as the sugar acceptor; the substrate concentration was set in a 0–400 μM range as the acceptor in Tris–HCl buffer (pH 7.0). For the acetylation of candidate AcBAHDs toward 1 and 12, 1 mM acetyl-CoA, 100 μM substrate, and 10 μg purified AcBAHD protein in 100 μL Tris–HCl solution  were incubated at 28 °C for 2 h. The reaction was terminated with an equal volume of methanol. Finally, the extracts were centrifuged for 10 min at 14,000 × g, and the catalytic products were analyzed by LC–MS.Full-length cDNA sequences of the E. coli BL21(DE3) expression strain. After incubating the bacterial culture, protein expression was induced by 1.0 mM IPTG for 22 h at 16 °C. The bacterial sediment was collected and resuspended in Tris–HCl (pH 6.5 for AcF6’H proteins or pH 8.0 for Ac4CL proteins). Protein purification was performed using the His-tag Protein Purification Kit .All AcF6′H and Ac4CL genes were then inserted into the pET-32a (+) vector and transformed into the 2, 0.03 mM CoA, and 2 mM substrate 13. To determine the kinetic values of Ac4CL1 and Ac4CL2, Substrate 13 at different concentrations ranging from 0.01 to 0.48 mM was added to the reaction mixture. Enzyme kinetic values were calculated from the concentration of 14 generated per minute using a molar extinction coefficient of ε = 18,000 M−1 cm−1.For the functional screen and kinetic assay to measure Ac4CL enzyme activity, spectrophotometry was used to measure the changes in absorbance values at wavelengths of 324 and 357 nm. Each 100 μL reaction mixture was prepared with a moderate quantity of Ac4CL protein, 50 mM Tris–HCl (pH 8.0), 0.5 mM adenosine 5′-triphosphate, 0.5 mM MgCl4, 1 mM disodium 2-oxoglutarate, 0.1 mM sodium ascorbate, and 14 at concentrations ranging from 0.005 to 0.3 mM to determine kinetic values and 2 mM to evaluate the candidate protein activity. The reaction was conducted at 30 °C for 2 min, 20 μL of 3 M NaOH was added, and the mixture was incubated at 37 °C for 10 min. Then, 20 μL of acetic acid was added, and the reaction mixture was centrifuged at 14,000 × g for 10 min. The supernatant was filtered through a 0.2 μm membrane filter and analyzed using LC–MS. Negative ion mode was used for product detection, and a C18 Waters Acquity UPLC® BEH C18 column  was used for chromatography, with flow phase A consisting of 0.1% formic acid and water and flow phase B consisting of acetonitrile. The flow rate was set at 0.3 mL/min, and the column temperature was maintained at 40 °C. The gradient program of the mobile phase was as follows: 5% B at 0 min; 5% B at 2 min; 6% B at 13 min; 95% B at 13.1 min, 95% B at 14.1 min; and 5% B at 14.5 min. To determine the kinetic values, a fluorescence marker was used to detect the fluorescence values at 345 nm/462 nm using an enzyme-linked immunosorbent assay. The enzyme kinetic parameters were calculated using GraphPad Prism 8.0.2.To conduct an enzyme assay with AcF6’Hs, each 100 μL reaction mixture was prepared with a moderate quantity of protein, 100 mM Tris–HCl (pH 6.5), 1.0 mg/mL BSA, 0.005 mM FeSOLC–QQQ–MS or LC–TOF–MS was used to analyze the catalytic products of AcUGTs, AcBAHDs, and AcF6’H. The LC–MS utilization and parameters are listed in Supplementary Table m/z range of 50–700 amu with a solvent delay of 8 min. 7, 8, and 9 were identified by comparing the retention time and mass spectra with the authentic standards 7, and the catalytic reaction products of BfCYP716Y1 and GmCYP72A69, respectively.For the identification of β-amyrin and corresponding hydroxylated products, GC–QQQ–MS analyses were performed on an Agilent Technology 7890 GC coupled with a 7000C Triple Quadrupole MS . One microliter of the sample was injected into splitless mode with an injector temperature of 250 °C. The column was DB-5 ms , 30 m × 0.25 mm i.d. × 0.25-μm film thickness, and was connected by a purged ultimate union (PUU) to provide sample separation. Helium was utilized as the carrier gas at a constant flow rate of 1.2 mL/min. The temperature was set as follows: start at 170 °C for 2 min, increase to 300 °C at 20 °C/min, and maintained at 300 °C for 11.5 min . The MS transfer line and ion source temperatures were set to 300 and 280 °C, respectively. Full mass spectra were generated by scanning within the 1 and 30 mM acetyl-CoA. A sufficient reaction was performed at 30 °C and 180 rpm for 5 h. Then, the reaction mixture was extracted three times with an equal volume of ethyl acetate, evaporated to dryness, and dissolved in 6 mL of ethyl acetate. Compound 11 was purified from this organic extract using preparative HPLC. The preparative HPLC was performed on an Agilent 1100 HPLC system equipped with a variable wavelength detector (VWD) and an evaporative light scattering detector (ELSD). The separation was performed on a 250 × 10 mm i.d. × 2.5 μm Cosmosil C18 column using water (A) and acetonitrile (B) at 2.5 mL/min with the following gradient: 0–55 min, 40% B; 56–60 min, 100% B; 61–70 min, 40% B. The injected volume was 100 μL, and the target product was collected between 21 and 23 min. The collected fractions were pooled and dried down through nitrogen blowing (to remove acetonitrile under a temperature of 0 °C) and freeze-dried (to remove water). This process yielded 1.5 mg of 11, further subjected to NMR in dimethylsulfoxide-d6. NMR spectra were recorded on a Bruker Avance Neo 600 MHz magnetic resonance spectrometer, with tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in parts per million (ppm) relative to the residual solvent.Bacterial cells expressing pET28a-AcBAHD6 recombinant proteins were harvested from 4 L of culture medium and resuspended in 480 mL of phosphate buffer. Crude enzymes were obtained by ultrasonication at 4 °C and then mixed with 50 mM 73. The RgTAL gene was amplified using pCS-TAL as the template. Plasmid pZE-649T was constructed by inserting AcF6’H1, Ac4CL2, AcUGT92G7, and RgTAL into pZE-12-luc using Kpn I, Sph I, BamH I, Not I, and Xho I. Information on the plasmids and the strains used is summarized in Supplementary Table 5, 1 mL of overnight seed cultures was inoculated into 50 mL of M9Y media supplemented with appropriate antibiotics. The strain BW1 was cultivated at 37 °C and 200 rpm for 3 h and then induced with 0.5 mM IPTG. The induced cultures continued to grow at 30 °C and 200 rpm. Samples were obtained every 24 h. Cell growth was monitored by measuring the optical density at 600 nm, and the concentrations of the product and the intermediates were analyzed by HPLC.The derivative plasmids pZE12-luc (high copy number) and pCS27 (medium copy number) were used for pathway assembly. The plasmids pCS-TPTA-HpaBC and pCS-TAL were constructed in our previous studyFurther 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 6Reporting SummarySource Data


Proteoglycans were purified from ovine gingival tissues and exposed to increasing H2O2 concentrations or a hydroxyl radical (·OH) flux for 1 h or 24 h, and ROS effects on proteoglycan core proteins and sulfated glycosaminoglycan (GAG) chains were assessed. ROS were capable of degrading gingival proteoglycans, with ·OH species inducing greater degradative effects than H2O2 alone. Degradative effects were particularly manifested as amino acid modification, core protein cleavage, and GAG chain depolymerization. Proteoglycan core proteins were more susceptible to degradation than GAG chains with H2O2 alone, although core proteins and GAG chains were both extensively degraded by ·OH species. Proteoglycan exposure to ·OH species for 24 h induced significant core protein amino acid modification, with decreases in glutamate, proline, isoleucine, and leucine; and concomitant increases in serine, glycine, and alanine residues. As clinical reports have previously highlighted proteoglycan core protein degradation during chronic gingivitis, whereas their sulfated GAG chains remain relatively intact, these findings potentially provide further evidence to implicate ROS in the pathogenesis of active gingivitis, complementing the enzymic mechanisms of periodontal tissue destruction already established.Reactive oxygen species (ROS) overproduction and oxidative stress are increasingly being implicated in the extracellular matrix (ECM) degradation associated with chronic inflammatory conditions, such as periodontal diseases. The present study investigated the effects of ROS exposure on the proteoglycans of gingival tissues, utilizing an  Porphyromonas gingivalis, are commonly associated with the initiation and progression of periodontal diseases [Periodontal diseases, comprising gingivitis and periodontitis, typically describe a range of inflammatory conditions which affect both the soft and hard tissues of the periodontium. They are regarded as the most common disease of mankind, leading to huge economic burdens for healthcare providers . As its diseases . Howeverdiseases . Consequdiseases ,7,9–11, diseases .2·−), hydrogen peroxide (H2O2), and the hydroxyl radical (·OH) species during periodontal diseases in response to periodontal pathogens and their products, or as a consequence of genetic disorders which alter neutrophil respiratory burst functions [Oxidative stress refers to the balance between ROS production and cellular/tissue antioxidant defense mechanisms. ROS are generated via a wide range of cellular mechanisms, although infiltrating neutrophils and macrophages are well-recognized as the principal source of superoxide radical  chain attached to a core protein. Numerous functions have now been attributed to the various proteoglycan species, including interaction with other ECM components, the sequestration of growth factors, the regulation of collagen fibrillogenesis; and in orchestrating cell attachment, migration, and proliferation responses, with these functions mediated via the core proteins or GAG chains . The pro2·−, H2O2 and ·OH) modify and degrade periodontal proteoglycans and GAGs, has demonstrated that the sulfated GAGs commonly found in periodontal tissues, such as chondroitin 4-sulfate, dermatan sulfate, and heparan sulfate, are more resistant to the modification of hexuronic acid and hexosamine residues and subsequent GAG chain depolymerization by ROS, compared to the high molecular weight, non-sulfated GAG, hyaluronan [in vitro, with proteoglycan degradation being particularly manifested as amino acid modification and core protein cleavage, with the GAG chains being less susceptible to ROS modification and degradation [in vitro [in vitro model system composed of supra-physiological (mM) oxidant concentrations beyond those expected in vivo [2O2 and ·OH, modify and degrade the core proteins and GAG chains comprising gingival proteoglycans; to ascertain whether the ROS-induced degradation of gingival proteoglycans contributes to the underlying mechanisms of ECM destruction associated with active gingivitis and periodontal diseases overall. Ovine gingiva was selected as a proteoglycan source, due to its availability and since periodontal disease in sheep (“broken mouth” disease) has previously provided a useful model for the study of periodontal disease pathology in humans [Therefore, it is apparent that the degradation of gingival proteoglycans during periodontal diseases would lead to a significant loss in structural integrity and altered functions in these tissues ,27. Our aluronan . Furtherradation . In contin vitro . Therefo in vivo ,47,48, tn humans ,50.n = 6), with relatively healthy dentition, obtained from a local abattoir. Gingival proteoglycans were isolated and purified by a modified method to that previously described for the isolation of proteoglycans from alveolar bone [Proteoglycans were isolated from ovine gingivae, dissected from the heads of 9-month-old Welsh Mule breed sheep  to remove any insoluble material, solubilized extracts were applied to a Q-Sepharose (HiLoad 16/10) anion exchange column incorporated into a Fast Performance Liquid Chromatography (FPLC) System  [The lyophilized ECM extract from the pooled gingivae was dissolved to a concentration of 10 mg/mL in 7 M urea (ThermoFisher Scientific), 0.05 M sodium acetate buffer, pH 6.8. Following centrifugation  ; and a 02O2 or to a ·OH flux, generated by the reaction of H2O2 and Fe3+ [2O2 for a prolonged period of 24 h. However, due to the highly reactive nature of ·OH species, gingival proteoglycans were subjected to a ·OH flux for 1 h and 24 h. Short and prolonged periods of exposure  were studied, in order to reflect the possible physiological conditions occurring during periodontal diseases.To examine the effects induced by exposing gingival proteoglycans to ROS, the purified proteoglycans were exposed to increasing concentrations of Hand Fe3+ ,51. Prot2O2 (ThermoFisher Scientific), was added at a final concentration of 20 mM or 60 mM. An ·OH flux was generated by the addition of H2O2 (60 mM) and FeCl3 . Reaction mixtures containing gingival proteoglycans (0.5 mg/mL) only; and gingival proteoglycans (0.5 mg/mL) with H2O2 (60 mM), FeCl3 (1.67 mM); and the established ·OH scavenger and transition metal ion chelator, thiourea , served as controls. Reaction mixtures containing H2O2 alone were incubated at 37 °C for 24 h only, whilst experimental and control reaction mixtures involving ·OH flux generation were incubated at 37 °C for 1 h and 24 h. For 1 h incubations, generated ·OH fluxes were terminated by the addition of the Fe3+ chelator, desferrioxamine mesylate , in 25 mM sodium acetate buffer, pH 5.6, containing 80 mM sodium chloride. Following incubation, reaction mixtures were lyophilized and dissolved in double-distilled water to a final proteoglycan concentration of 15 mg/mL. Reaction mixtures were stored at −20 °C until required for analysis.Reaction mixtures , were established containing gingival proteoglycans at a final concentration of 0.5 mg/mL in 25 mM sodium acetate buffer, pH 5.6, containing 80 mM sodium chloride. To each reaction mixture, Hn = 3 independent occasions for each experimental group.Gel filtration chromatography was performed to assess the fragmentation of the gingival proteoglycans to lower molecular weight products when subjected to ROS over 1 h and 24 h. Aliquots (25 µL) of each reaction mixture were applied to a Superdex 75HR 10/30 column (fractionation range 3–70 kDa), incorporated into the FPLC System; and eluted with 2 M guanidinium chloride, 0.5 M sodium acetate buffer, pH 6.8, containing 0.35 M sodium chloride . Fraction = 3 independent occasions.Cellulose acetate electrophoresis was performed in order to qualitatively and quantitatively determine the GAG constituents of the gingival proteoglycans and the effects of ROS exposure upon GAG content. Aliquots (2 µL) of each reaction mixture were applied to cellulose diacetate sheets  in 0.2 M calcium acetate buffer (Sigma), pH 7.2; and separated at 0.6 mA per cm width of the sheet for 4.5 h ,53. A coAmino acid analysis was performed to qualitatively and quantitatively determine the amino acid constituents of the gingival proteoglycan core proteins and to identify those amino acids susceptible to modification by ROS. Following the incubation of experimental and control reaction mixtures at 37 °C for 24 h, aliquots (100 µL) of each reaction mixture were desalted using PD-10 columns , using double-distilled water as an eluent and lyophilized. Samples were hydrolyzed in 6 M HCl , under vacuum at 108 °C for 18 h. The separation and characterization of the individual amino acids were performed on a Pickering cation exchange amino acid analysis column, integrated into a High Performance Liquid Chromatography (HPLC) System . Amino acids were detected by the reaction of post-column eluent with ninhydrin at 55 °C and the absorbance was measured at 540 nm. Amino acid concentrations were calculated by comparison with the absorbance values obtained from a standard amino acid mixture for protein hydrolysates (Sigma) . Amino an = 3 independent occasions, in triplicate. Statistical analyses were performed using GraphPad . Data were expressed as mean ± standard deviation (SD) and statistically compared using Analysis of Variance (ANOVA), with post-hoc Tukey test. Statistical significance was considered at p < 0.05.The quantification of % GAG loss following ROS exposure was performed on Supplementary Figure 1(A)), with fraction III being identified as the proteoglycan-rich fraction for the isolated gingival extracts, as determined following cellulose acetate electrophoretic separation and Alcian blue staining ). Four major fractions (I–IV) were also resolved following the further purification of the partially purified gingival proteoglycan extracts by Resource Q anion exchange chromatography ), with fraction III again being recognized as the proteoglycan-rich fraction, following cellulose acetate electrophoretic separation and Alcian blue staining ).The isolation and purification procedures described yielded proteoglycan extracts associated with the ECM of pooled gingival tissues. Typical 280 nm elution profiles for the sequential separation and purification of proteoglycans from gingival tissues by Q-Sepharose (HiLoad 16/10) and Resource Q anion exchange chromatography are shown in  and S2(A)), respectively. The elution profiles obtained were typical of those previously reported for the purification of proteoglycans from other oral tissues, such as alveolar bone, dentin, and dental pulp ,53. FromoV, tV) of the column, for the gingival proteoglycans exposed to the ·OH flux , compared to untreated gingival proteoglycans  and ∼13% loss following 60 mM H2O2 (p < 0.001) treatments over 24 h, compared to untreated controls , although GAG loss was still significant compared to untreated gingival proteoglycans , to promote amino acid modification and cleavage of the core proteins of the large aggregating, chondroitin sulfate/keratan sulfate cartilage proteoglycan, aggrecan, without extensive sulfated GAG chain depolymerization [2O2 and ·OH treatments have also been demonstrated with the chondroitin sulfate-substituted SLRPs in alveolar bone, decorin and biglycan [−) [2O2 alone, compared to sulfated GAG chains. However, both the core proteins and GAG chains are highly susceptible to degradation by ·OH species, especially following prolonged exposure.Detailed analysis of the respective core protein and GAG chain constituents of gingival proteoglycans following ROS treatment, determined that the gingival proteoglycans remained relatively intact following exposure to Hrization ,48,54,55biglycan ; in addiycan [−) ,57. Howeycan [−) ,48,54–57Much research has been performed into elucidating the precise mechanisms and oxidative products induced by ·OH species on GAG chain structures . SulfateThe hypothesis that core protein modifications by ROS were particularly responsible for decreases in gingival proteoglycan molecular size, was supported by amino acid analysis. The extensive degradation of gingival proteoglycans following prolonged ·OH flux exposure had a prominent effect on the amino acid composition of the core proteins. The amino acid analysis identified major reductions in glutamate, proline, isoleucine, and leucine contents, with corresponding increases in the detectable levels of serine, glycine, and alanine residues. Such findings suggested that glutamate, proline, isoleucine, and leucine were the most susceptible to modification and degradation by an ·OH flux. Of particular interest was the loss of leucine residues, as SLRPs, decorin, biglycan, lumican, and fibromodulin, all contain leucine-rich repeat sequences which are implicated in the orchestration of specific functions, such as collagen fibrillogenesis ,29.2O2 exposure, the elevated levels of serine, glycine, and alanine residues detected imply that a degree of amino acid modification had occurred with H2O2 treatment alone. Therefore, such conclusions reinforce the concept that highly susceptible amino acid residues within the core protein structures of gingival proteoglycans, such as glutamate, proline, isoleucine, and leucine, are the prominent regions of peptide bond cleavage and protein degradation following ROS exposure.Proteins are well-established to be highly susceptible to ROS-induced damage, mediated via amino acid modification which leads to increased cross-linking, aggregation, peptide bond cleavage, and fragmentation; which ultimately influences protein structure and functions ,65. Amin2O2 alone compared to other sulfur-containing amino acids, such as cysteine; with susceptibility dependent on specific ROS encountered [2O2-treated samples may partly be a consequence of technique or data analysis sensitivity. However, as evident herein, previous studies have shown that methionine residue levels are maintained in the core proteins of alveolar bone proteoglycan exposed to H2O2 alone, although methionine residues are undetectable due to extensive modification following ·OH exposure, via H2O2/Fe3+ generation [2O2 and ·OH fluxes [Intriguingly, however, there were certain proteoglycan core protein amino acids, such as methionine, histidine, and tyrosine, whose susceptibilities to ROS-induced modification are well-recognized, but which did not exhibit extensive residue loss upon ROS exposure. Methionine oxidation is reported to be much slower when exposed to Hountered ,59. In lountered , gingivaneration . SimilarH fluxes , despiteH fluxes ,59. HoweH fluxes , similarH fluxes .in vitro model system composed of supra-physiological (mM) oxidant concentrations, in order to study the mechanisms by which ROS modify and degrade gingival proteoglycans. Similar approaches have previously proven beneficial in elucidating the mechanisms by which ROS modify and degrade proteoglycans (at mg/mL concentrations) purified from various other tissues, including cartilage [2O2 and Fe3+ concentrations implemented herein to induce proteoglycan modification/degradation can be regarded as being beyond those expected in vivo within the tissues and biofluids of the oral cavity which would limit the physiological significance of our findings, for instance, compared with the μM H2O2 concentrations detectable in saliva [As alluded to above, the present study has utilized an artilage ,48 and aartilage . Thus, tn saliva ,67. Nonen saliva ,47,48.Despite the present study indicating that gingival proteoglycan core proteins are predominantly affected by ROS, even minor changes induced by ROS within the GAG chain structures could further have potentially detrimental consequences on the architecture, integrity, and functionality of the gingival ECM. Indeed, there are ever-increasing numbers of core protein- and GAG chain-mediated functions attributed to prominent gingival proteoglycans, decorin, versican, and syndecans, including the modulation of ECM component interactions, coordinating collagen fibrillogenesis, growth factor sequestration; and in the regulation of inflammation, cell signaling, attachment, migration, and proliferation ,29. ThusNumerous studies have demonstrated the detrimental effects of active periodontal disease on the gingival ECM. Gingival connective tissue-associated, decorin and biglycan, have both been demonstrated to be markedly depleted in chronically inflamed periodontal tissues , with th