We report on the first examples of isolated silanol–silanolate anions, obtained by utilizing weakly coordinating phosphazenium counterions. The silanolate anions were synthesized from the recently published phosphazenium hydroxide hydrate salt with siloxanes. The silanol–silanolate anions are postulated intermediates in the hydroxide‐mediated polymerization of aryl and alkyl siloxanes. The silanolate anions are strong nucleophiles because of the weakly coordinating character of the phosphazenium cation, which is perceptible in their activity in polysiloxane depolymerization. Silanol–silanolate anions that are devoid of any contacts to the counterion were synthesized by treatment of siloxane species with a phosphazenium hydroxide hydrate salt. Because of the weakly coordinating cation, the silanolate anions exhibit strong silanol–silanolate hydrogen bonding. The [D3OH]− salt was successfully tested in the depolymerization of polydimethylsiloxane with an outstanding activity relative to NaOH or KOH. Silicones constitute the passage between organic and inorganic polymers with outstanding chemical and physical properties and are of broad scientific and industrial interest.4). The latter can be converted into linear polysiloxanes by ionic ring‐opening polymerization reactions.The intermediately formed α,ω‐siloxanediols eliminate water under formation of the final silicones or cyclic siloxanes like octamethylcyclotetrasiloxane D. The lat2O]nH, form inter‐ and intramolecular hydrogen bonds that often result in the formation of ring structures 2]2−, with metal halides under several reaction conditions, which resulted in the formation of six‐ and eight‐membered metallacyclosiloxanes.Anionic silanolates can be synthesized by cleavage of siloxanes with strong bases.Regarding the chemical robustness of silicones in nature, their multi‐ton production causes serious waste disposal problems. The degradation of siloxanes under formation of cyclic derivatives is of general interest because it forms the basis of a possible recycling of widely used silicone plastics.The depolymerization of silicones can be achieved under acidic, basic, and fluorinating conditions, leading to low‐molecular cyclic siloxanes or organosilyl fluorides, which can be converted into new silicone plastics afterwards.1H][OH(OH2)n]  and 329.0(1) ppm; both distances are longer than the sum of the van der Waals radii. The donor hydrogen atom is disordered as well, and bonded to O1 or O3. Both positions were refined isotropically, but restrained to have the same O−H distances.Compound 3 exhibit an O1–O2 distance of 258.2(1) pm and an O1–O3 distance of 252.4(5) pm.Compared to the O–O distances of the hydrogen bridges in the triphenylsilanol pyrrolidine complex of Strohmann and co‐workers ,1 and 2, silanolate salt 3 undergoes rapid decomposition in H‐ and C−H‐acidic solvents and requires handling in chlorobenzene solution. A 31P and 29Si{1H} NMR spectroscopic investigation of the dissolved product 3 indicated the presence of the protonated phosphazene +[1H] and also a reformation of Me3SiOSiMe3, which points to the equilibrium reaction shown in Scheme 29Si{1H} NMR spectrum at δ=−8.6 ppm was attributed to the silicon atoms of the silanolate anion in 3. As observed for the hydroxide hydrate salt 2, silanolate salt 3 decomposes in vacuum by the deprotonation of its cation +[1H] and liberation of silanol and disiloxane.2 and the silanolate anion in 3 . After the addition of water, the reaction with the in situ generated hydroxide produced a second phase. In the upper phase, a mixture of cyclic compounds, mainly D4 and D5 and traces of D3, were detected by 1H–29Si HMBC NMR spectroscopy. This result clearly underlines the existence of a fast equilibrium between cyclic species, whose interconversion is catalyzed by 2. The initial ring‐opening reaction is presented in Scheme 31P NMR spectroscopic analysis confirmed that there are no phosphorus species in the upper phase.In the following, phosphazene 4 were obtained in an excellent yield of 95 %. X‐ray crystallographic analysis3OH]− in 4, which forms an intramolecular hydrogen bridge in the solid state  pm is observed between O1 and C31. The disordered donor hydrogen atom was refined isotropically at two positions with a ratio of 1:1; both were restrained to have the same distances to oxygen atoms O1 and O4, respectively.4 (Si‐O distances from 164 to 166 pm).Based on potentiometric titrations of siloxanes, the thermodynamically favored eight‐membered‐ring structure was already proposed by Baney and Atkari in 1967.4 could not be determined in the IR analysis. The 1H NMR spectrum in C6D6 displayed a broad singlet at δ=14.0 ppm, which was assigned to the proton involved in the hydrogen bridging. In the 29Si{1H} NMR spectrum of 4 the signal for the silicon atoms adjacent to the hydrogen bridge was observed at δ=−23.9 ppm.The O‐H‐O vibration mode of 4 is also accessible from the treatment of D4 or D5 with the phosphazenium hydroxide, as evident by NMR and X‐ray analysis. The reaction of 2 with equimolar quantities of D5 afforded compound 4 in a 85 % yield. A plausible reaction pathway mirrors a series of equilibria and confirms the high thermodynamic stability of the hydrogen‐bridged eight‐membered ring.Compound 4 begins to decompose slowly at 90 °C in vacuo, with fast decomposition above 100 °C. The volatile products were identified by GC‐MS analysis as water and cyclic siloxanes, with D4 as the main component. A 31P NMR spectroscopic investigation of the residue revealed the free phosphazene 1, and the decomposition route is thus similar to that of hydroxide hydrate 2.Silanolate salt Ph23OH]− anion in salt 5, which was isolated in excellent yield  and were subjected to X‐ray crystallography  ppm shows the same value as in 2, as well as the silanolate anions in 3, 4, and 5 exhibit increased nucleophilicity. This was exemplary shown for silanolate salt 4 in the depolymerization reaction of a polydimethylsiloxane with terminal trimethylsilyl groups  with an average rate of 3.1(2) mL h−1.4 was regained as a colorless solid devoid of any traces of decomposition. The obtained distillate consisted mainly of D4 (82 %) as well as D5 (8 %) and traces of D3 .The equilibrium activity can be determined by the temporal quantity of cyclic siloxane species, which are removed from the siloxane mixture by distillation. The silanolate salt 4 in silicone oil was formed, whereby the averaged distillation rate of volatiles was determined to be 24.7(19) mL h−1. The average composition of the distillate does not significantly differ from that of the run at room temperature (Scheme −1) or potassium hydroxide (<1 mL h−1) mediated depolymerizations with concentrations of 11 mol % carried out at 90 °C, compound 4 showed a significantly enhanced equilibrium activity (Table S1 in the Supporting Information).As industrial processes are preferably run at higher temperatures and higher pressures, we also mimicked these conditions by applying a membrane pump vacuum (7 mbar) and a temperature of 90 °C. Under these conditions, a clear solution of e Scheme . In comp2)n]− in 2 in the presence of the weakly coordinating phosphazenium cation +[1H]. The silanolate anions are not in contact with the counterion, and the [D3OH]− salt 4 and the [DPh23OH]− salt 5 show only intramolecular hydrogen bonding. Similar to the hydroxide hydrate salt 2, trimethylsilanolate salt 3 decomposes in vacuum. An NMR spectroscopic investigation provided evidence for an equilibrium reaction of hydroxide salt 2 and silanolate salt 3. The salts [1H][D3OH] (4) and [1H][DPh23OH] (5) were synthesized in excellent yields of over 95 % and structurally characterized.In conclusion, we have reported on the first three silanol–silanolate anions in the condensed phase, which were synthesized by the reaction of the in situ generated hydroxide hydrate anion [OH should be addressed to the authors.SupplementaryClick here for additional data file.


Accumulating studies identified that BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) is integrally involved in the initiation and development of tumors. Nevertheless, the precise biological role and underlying mechanisms of BUB1B in hepatocellular carcinoma (HCC) remain indistinct.To figure out the role of BUB1B in HCC, we first assessed its expression using The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) databases. We then verified BUB1B expression in HCC tissues, nontumor tissues, and HCC cell lines through western blotting, quantitative reverse transcription‐polymerase chain reaction, and immunohistochemistry. To explore the specific function of BUB1B in HCC in vivo and in vitro, we performed the flow cytometry, Cell Counting Kit‐8, 5‐ethynyl‐2′‐deoxyuridine incorporation, colony formation, Transwell, wound‐healing, subcutaneous tumor growth, and metastasis assays. Additionally, we identified the BUB1B‐regulated pathways involved in HCC by using gene set enrichment analysis.Our data displayed that higher BUB1B expression was detected in HCC tissues and HCC cell lines. The overexpression of BUB1B was positively correlated with adverse clinicopathological characteristics. Survival analyses showed that lower recurrence‐free and overall survival rates were correlated with the overexpression of BUB1B in patients with HCC. Moreover, the malignancy of HCC was facilitated by BUB1B both in vivo and in vitro. Lastly, the results were confirmed by western blots, which showed that BUB1B upregulated mTORC1 signaling pathway in HCC. Meanwhile, the oncogenic effect of BUB1B will be impaired when the mTORC1 signaling pathway was inhibited by rapamycin.We highlighted that BUB1B played an oncogenic role in HCC and was identified as a possible clinical prognostic factor and a potential novel therapeutic target for HCC. Our study confirmed that BUB1B is overexpressed in HCC tissues and cell lines, and the overexpression of BUB1B is highly correlated to adverse clinicopathological features, lower recurrence‐free survival and overall survival rate in patients with HCC. Our results revealed the oncogenic role of BUB1B in HCC through a series of functional assays. Moreover, we identified the BUB1B/mTORC1 signaling axis as a critical regulator of the progression in HCC. This is especially true for Asia where unfortunately most patients are from China.Bub1b , encoded by BUB1B, belongs to the core elements of the spindle assembly checkpoint (SAC), which includes Mad1, Mad2, Mad3/Bub1b, Mps1, Bub1, and Bub3.Our present study demonstrates that BUB1B is upregulated in HCC tissues and HCC cell lines compared to adjacent normal liver tissues and normal hepatic cell line, respectively. Survival analyses uncovered that the overexpression of BUB1B owned a positive relation with a shorter recurrence‐free rate and worse overall survival in patients suffering from HCC. BUB1B contributed to the HCC progression by facilitating the proliferation, migration, invasion, epithelial‐mesenchymal transition (EMT), metastases, inhibiting cell apoptosis, and lowering G0/G1 phase arrest of HCC cell lines. Finally, the mTORC1 signaling pathway upregulated by BUB1B was detected, as indicated by gene set enrichment analysis (GSEA). Altogether, our research findings demonstrate that BUB1B is a likely clinical prognostic biomarker and a novel therapeutic target for HCC.22.1We obtained human liver tissue samples consisting of the tumor tissues and adjacent normal liver tissues from 80 patients suffering from HCC who underwent liver partial hepatectomy at the Hepatobiliary Center of the First Affiliated Hospital of Nanjing Medical University. All tissue samples were partly stored in formaldehyde solution and the rest was directly frozen in liquid nitrogen for storage before executing experiments. Our study obtained the consent of the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University before the research began.2.The six human HCC cell lines  and the normal hepatic cell line L02 were purchased from the American Type Culture Collection . We cultured all cells with Dulbecco's modified Eagle's medium  with the addition of 10% fetal bovine serum (FBS) and 1% antibiotics  at 37°C in a humidified incubator containing 5% CO2.2Lentiviral vectors encoding BUB1B (LV‐BUB1B), short‐hairpin RNAs (shRNAs) against BUB1B (LV‐shBUB1B), and corresponding empty vectors (LV‐control/LV‐shNC) were obtained from GenePharma Biotech . The infection efficiency was enhanced by adding 5 μg/ml polybrene (Genepharma) to the cell lines.The stably transfected cell lines were selected by adding 6 μg/ml of puromycin  into the cells for 7 days. In the rescue experiments, we treated the LV‐BUB1B‐transfected HepG2 cells with rapamycin (100 nmol/L).2.3The Cell Counting Kit  was utilized to assess cellular proliferative potential on the basis of the manufacturer's protocol. In brief, we planted the stable cell lines into 96‐well plates (500 cells/well) with the addition of CCK‐8 solution (10 μl/well). After incubation for 2 hours in the incubator described as above, we measured the absorbance (450 nm) of each well.2.4We seeded the stable cell lines into 6‐well plates (500 cells/well). After culturing all cells with DMEM for 14 days, we discarded the culture media and washed the cells with PBS two times. Then we fixed the cells with 75% ethyl alcohol for 5 min. Finally, we identified the proliferating colonies by staining them with crystal violet dye solution  after rinsing with PBS three times.2.5g at 4°C for 5 min, we washed the cells with precooled PBS two times and then pelleted the cells through repeat centrifugation. We collected the cell sediment and resuspended them with 100 μl binding buffer, subsequently added 5 μl FITC‐conjugated Annexin V and 5 μl PI into the cell suspension. At room temperature the reaction lasted for 10 min, during which time it needs to be completely in the dark. At last, the cell suspension was supplemented with 400 μl binding buffer, then the mixture was gently blended. The apoptosis level of cell samples would be detected by the flow cytometry within 1 hour.We made use of the Annexin V‐FITC/propidium iodide (PI) Apoptosis Detection Kit  to identify apoptotic cells on the basis of the manufacture's protocol. After dissociating the stable cell lines with trypsin without EDTA and centrifuging at 300g for 5 min. After rinsing the cell lines with PBS and repeating the centrifugation as above, we discarded the PBS and first fixed the cell lines with 4 ml of 75% ethyl alcohol for 30 min, and then stored them at −20°C overnight. The next day, we collected the cell lines after centrifugation at 400 g for 5 min, then rinsed them with PBS two times and repeated the centrifugation as above. For cell cycle analysis, it was required that the cells were incubated with 50 mg/ml RNase and 500 μl PI solution for 20 min at room temperature, during which time it needs to be completely in the dark. The data on apoptosis and cell cycle were analyzed by the FlowJo software (Tree Star Inc).For the cell cycle assay, we utilized a Cell Cycle Analysis Kit . We first digested the stable cell lines with trypsin for 2 min, then added DMEM to the culture dish to stop the digestion. We collected the cell suspension and centrifuged them at 400 2.6The Transwell chambers  were utilized to evaluate the migratory and invasive capabilities of the cells. The main difference between cell invasion and migration assays is that the Transwell plates (8‐µm pore size) were coated with Matrige for the former and left uncoated for the latter. We planted the cell lines into the upper chamber and cultured them with 200 μl DMEM with no addition of FBS; however, we filled the lower chamber with 500 μl complete medium containing 10% FBS, which attracted the cells across the Transwell membrane into the lower chamber. After one‐day incubation in the incubator described as above, we discarded the culture medium in the upper chamber and washed the Transwell chambers with PBS two times, then fixed the cells with methyl alcohol for 30 min. The invasive or migratory cells were dyed with 0.1% crystal violet for 30 min in the dark at room temperature. The chambers were rinsed with PBS three times and the stained cells were observed through a light microscope.2.75 cells/well) into 6‐well plates. After about 48 hours of culture, the cells almost reached confluency. After washing away nonadherent cells two times with PBS, a uniform linear scratch was made using a sterile 200‐μl pipet tip in the center of the well. Taking the distance between the wound edges at 0 hour after rinsing two times with PBS as a baseline, we acquired the image of the same location after 48 hour. The change in the distance was measured to assess the cellular ability of migration and repair.We planted the SMMC7721 and HepG2 cell lines  and RNase‐free ddH2O.We utilized the EasyPure RNA Kit (Ruijie Biotech) to extract total RNA from tissue and cell samples on the basis of the manufacturer's instructions. In short, tissues or cells were mixed with the lysis buffer. After the efficient lysis was confirmed, the lysis solution was collected and vortexed for 10 sec, then added with an equal volume of absolute ethyl alcohol and mixed together. The lysis solution was transferred into an RNA column, which was placed in a centrifuge tube. The tube was centrifuged at 4000TM Universal SYBR qPCR Master Mix  based on the standard protocols. Primer sequences used in this study for PCR amplification were as follows: BUB1B: 5′‐CTTAGGGTGCAGCTGGATGT‐3′ (forward) and 5′‐ACCCATCCCAGAAGACCTGT‐3′ (reverse); β‐actin: 5′‐TGACGTGGACATCCGCAAAG‐3′ (forward) and 5′‐CTGGAAGGTGGACAGCGAGG‐3′ (reverse). See We performed the qRT‐PCR with the ChamQ2.9We used the whole protein extraction kit (KeyGEN BioTECH) to extract tissues and cell proteins on the basis of the manufacturer's instructions. All protein samples were separated by 10% SDS‐PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. The antibodies used were listed as follows: BUB1B (Abcam), E‐cadherin, N‐cadherin, Vimentin, Bax, cleaved caspase 3, Bcl‐2, CDK2, CDK4, CDK6, mTOR, S6, p‐S6 (at Ser240/244), P70S6K, p‐P70S6K (at Thr389), p‐mTOR (at Ser2448), GAPDH, horseradish peroxidase (HRP)‐conjugated anti‐rabbit IgG antibodies . See 2.10Once the samples were obtained, we immediately fixed them in 4% formaldehyde solution, then embedded them in the paraffin. We cut the paraffin block into 4‐μm slices and incubated them with primary antibodies for BUB1B and Ki‐67 (Abcam) for 12h at 4°C.After that, we incubated the slices with secondary antibody conjugated with HRP for 1h at room temperature. Finally, we stained the slices with 3,3′‐diaminobenzidine and hematoxylin for detection. Two independent pathologists in a blind study assessed the positivity and intensity of the staining. Ten fields from each slide were selected randomly for examination.2.114 cells/well) and cultured with the complete medium for 1 day. Subsequently, the cell lines were incubated with 50 μmol/L EdU (RiboBio) for 2 hours at 37°C. Then, we fixed the cell lines with 4% formaldehyde solution for 30 min. Subsequently, the cell lines were permeabilized with 0.5% TritonX‐100 for 10 min. We added 400 μl 1 × ApolloR reaction cocktail and subsequent 400 μl Hoechest33342 to visualize the EdU‐positive cells after rinsing the cell lines with PBS three times. At last, we obtained the images of cells through a microscope.The EdU assay kit  was used to assess cellular proliferation capacity in the cell lines, on the basis of the manufacturer's instructions. In short, the stable cell lines were planted into 96‐well plates  on the basis of the manufacturer's instructions. Ten fields from each slide were selected randomly for examination.2.11.26 cells) resuspended in 100 μl PBS were injected subcutaneously into the nude mice (n = 6 per group) for subcutaneous tumor growth assays. For metastasis assays, 1 × 106 of HCC cells expressing luciferase were injected into the caudal vein of nude mice (n = 10 per group). After 5 weeks, the occurrence of distant metastases was observed using the IVIS 100 Imaging System .In this study, all procedures involving animals were full of humanistic care and obtained the consent of the Institutional Animal Care and Use Committee of the Nanjing Medical University before the research began. We purchased 4‐week‐old female BALB/c nude mice from the Department of Laboratory Animal Center of Nanjing Medical University. The stably transfected cell lines . All experiments were repeated at least three times and the differences deemed statistically significant when p‐value < 0.05.All data in this study are expressed as mean ± SD. We made use of the SPSS software ver. 20.0 and GraphPad Prism 8.0 to analyze the data and calculate the 33.1To determine the expression pattern of BUB1B in HCC, we downloaded RNA sequencing data from TCGA (371 HCC tissues and 50 nontumor tissues). The data displayed that the expression of BUB1B was markedly upregulated in HCC tissues than the normal tissues Figure . In line3.2Based on the western blotting and qRT‐PCR results shown in Figure First, the relationship between BUB1B and the proliferating cell nuclear antigen (PCNA) was assessed. We noticed a positive relation between the expression of BUB1B and PCNA Figure . The CCK3.3Rate of apoptosis and cell cycle alteration are two important factors in the progression and development of HCC. We performed the flow cytometry experiments to investigate the rate of apoptosis and the cell cycle profile. Our results displayed that the knockdown of BUB1B promoted the apoptosis of SMMC7721 cells, while overexpression of BUB1B in HepG2 cells reduced cellular apoptosis Figure . MoreoveWe then investigated whether BUB1B influenced the cell cycle of HCC cells. The results displayed that the downregulation of BUB1B increased the percentage of G0/G1 phase in the SMMC7721 cells, as demonstrated by the cell cycle assay Figure . We obse3.4We generated subcutaneous xenograft tumor mouse models of HCC to further examine the effect of BUB1B on tumor progression. Tumors from mice injected with SMMC7721‐sh1 cells showed the slower growth and lower average tumor volume and weight Figure . Convers3.5To further determine the potential signaling pathways modulated by BUB1B, we performed GSEA via analyzing the data from TCGA. We observed that the activity of the mTORC1 signaling pathway was positively correlated with BUB1B expression levels in 371 HCC tissues Figure . We dete4P = .00072) and worse overall survival . Given the clinicopathologic significance of BUB1B expression that is associated with HCC progression and poorly prognosis, we evaluated the function of BUB1B in the malignancy of HCC cell lines both in vivo and in vitro. Functional analysis demonstrated that the upregulation of BUB1B contributed to the proliferation, migration, invasion, and metastases of HCC cells. It is widely known that EMT is highly involved in HCC progression and metastases.Recent studies have shown that BUB1B has a variety of effects on the initiation and progression of multiple tumors. It has been reported that the loss of acetylation of the Bub1b results in defects in spindle assembly checkpoint signaling and promotes tumor formation.BUB1B was identified as an oncogene in prostate cancer through BUB1B‐dependent regulation of MELK transcription.It is well known that the serine/threonine kinase mammalian target of rapamycin (mTOR) is a pivotal kinase that governs cell proliferation, growth, and survival under diverse environmental stimuli, including oxygen, amino acids, energy, growth factors, and stress.In summary, our study confirms that BUB1B is overexpressed in HCC tissues and cell lines. The overexpression of BUB1B is highly correlated with adverse clinicopathological features, lower recurrence‐free survival, and overall survival rate in patients suffering from HCC. Our results revealed the oncogenic role of BUB1B in HCC through a series of functional assays. Moreover, we identified the BUB1B/mTORC1 signaling axis as a critical regulator of the progression in HCC. Future research should focus on the BUB1B/mTORC1 signaling axis, which can serve as a prognostic biomarker and possibly offer a novel therapeutic strategy for the treatment of HCC.All authors of this study confirm that they have absolutely no conflict of interest concerning this manuscript.LL, JR, and JQ conceived of the project and designed the research. JQ, SZ, PW, HW, BS, HP, and ZJ carried out experiments in vivo and vitro. JQ accomplished the manuscript. SZ and PW performed the data analysis. LL and JR offered a lot of suggestions. LL and JQ revised the manuscript. All the authors read and approved the final version to be published.Supplementary Materials and MethodsClick here for additional data file.


Most of computational methods of building RNA tertiary structure are template-based. The template-based methods usually can give more accurate 3D structures due to the use of native 3D templates, but they cannot work if the 3D templates are not available. So, a more complete library of the native 3D templates is very important for this type of methods. 3dRNA is a template-based method for building RNA tertiary structure previously proposed by us. In this paper we report improved 3D template libraries of 3dRNA by using two different schemes that give two libraries 3dRNA_Lib1 and 3dRNA_Lib2. These libraries expand the original one by nearly ten times. Benchmark shows that they can significantly increase the accuracy of 3dRNA, especially in building complex and large RNA 3D structures. Compared with the old library , we mainly made the following improvements: (1) To enrich templates in the library, instead of retaining only non-homologous RNA monomer structure like in 3dRNA_Oldlib, the chains from different RNAs with the same sequence and secondary structure are reserved. (2) All modified nucleotides are retained by mutating them into standard ones. (3) All base pairs  calculated by X3DNA 2The 3D template library of 3dRNA is constructed by decomposing RNA molecules with known 3D structures into SSEs. The SSEs are defined as stem and different kinds of loops together with two base pairs of each stem connected with them, see . The looThe construction of the 3D template library mainly includes PDB filtering and chain splitting, secondary structure calculation and SSE module decomposition. The detailed construction process of the 3D template library is shown in PDB filtering and chain splitting. We searched the RCSB PDB database Secondary structure calculation. In order to decompose these 11,756 RNAs into SSEs, we also need to obtain their secondary structures. Here X3DNA is used to do this. Furthermore, we retain not only the standard base pairs  but also all non-standard base pairs. Single-stranded RNAs without secondary structures are removed and for identical strands one of them is retained. Finally, 6,409 RNA monomers remained in this step.SSE decomposition. According to the secondary structures of 6409 RNA monomers, their 3D structures are split into 3D templates according to the SSEs. These templates and their related information are put into the template library of 3dRNA. In order to facilitate the template searching in the library, the relevant information of the 3D templates of each SSE includes its sequence, dot-bracket notation, length and family. The decomposition adapts two schemes and so results in two different libraries: 3dRNA_Lib1 and 3dRNA_Lib2. In 3dRNA_Lib1 single base pair will be opened as in 3dRNA_Oldlib. This will decrease the accuracy of SSE secondary structure but increase the tolerance in template selection. In 3dRNA_Lib2 single base pair will be preserved to ensure the accuracy of SSE secondary structure. The final numbers of helices and loops in the template library 3dRNA_Lib1 and 3dRNA_Lib2 are the same, except the dot-bracket representations of the secondary structure of the SSEs in which single base pair is opened in 3dRNA_Lib1.As an example, 3In comparison with about 50,000 templates in 3dRNA_Oldlib, the library 3dRNA_Lib1 has now been expanded by about 10 times. 3.1In order to verify the correctness of our template library construction and test the overall prediction performance of 3dRNA with the new template libraries, all RNAs (6409 single-strand RNAs) are used as a test set (Test Set I). Since the optimization of 3dRNA is very time-consuming, only assembled structures are given here. We first use “3dRNA_Lib1” and “3dRNA_Lib2” to predict 3D structures of the RNAs in Test Set I with self-inclusion to see whether the SSEs of each RNA can find themselves as their templates in the 3D template libraries. The prediction results in “3dRNA_Lib1” are shown in 3.2In order to test the performance of the improved template library in predicting short RNAs, we analyzed 32 RNA used by 3dRNA-2.0 In general, the average RMSD of the assembled structures is reduced from 5.19 Å of “3dRNA_Oldlib” to 3.19 Å of “3dRNA_Lib1”, and that of the optimized structures is reduced from 4.16 Å of “3dRNA_Oldlib” to 3.03 Å of “3dRNA_Lib1”. In particular, the poor predictions for 1NYI_1, 28SP_0, 1J1U_0, 1N8X_0, and 1Z43_0 with “3dRNA_Oldlib” are obviously improved with “3dRNA_Lib1”. The prediction accuracies of “3dRNA_Lib1” and “3dRNA_Lib2” are similar in this case.3.3In order to show the advantages of the new template library more intuitively, we have collected some RNAs that are very difficult to predict using “3dRNA_Oldlib” and name them as Test Set III. This test set includes 21 RNAs of different types and with lengths between 28nt to 158nt. The detailed description of them is shown in In order to have a fair comparison with RNAComposer, both the lowest RMSD B and the3.4As mentioned above, at present, it is quite difficult to predict the 3D structures of long RNAs. We found that 3dRNA with the new template library can significantly improve the accuracy of predictions for long RNAs. We selected 5 large riboswitches, ranging from 500nt to 3000nt and name them as Test Set IV to show this. Among the five RNAs, 3dRNA using 3dRNA_Oldlib can only predict the structures of 1C2W_0 and 1FFZ_0 due to the limitation of the library. Not only can “3dRNA_Lib1” and “3dRNA_Lib2” predict the 3D structures of the five RNAs, but the assembled structures of four of them have good performance with the accuracy within 15 Å. RNAComposer can only predict one of the five RNAs within 500nt (1FFZ_0). 3.5Here 12 challenges of RNA-Puzzles We also used RNAComposer to predict these 12 puzzles with the same 2D structures. Overall, the prediction accuracy of RNAComposer is similar to that of “3dRNA_Oldlib”. The puzzle5, puzzle6, puzzle8, puzzle13, puzzle14, and puzzle21 have very poor prediction results due to the lack of templates for the corresponding loop regions in FRABASE 3.6In order to estimate the consuming time of assembling 3D structure by 3dRNA, the running times of 588 RNAs in Test Set I are given in 4We have improved the 3D template library of 3dRNA. With the number of solved RNA 3D structures increasing in the PDB, the templates of 3dRNA will be continuously enriched. Comparing with the previous template library, the number of the templates in the improved template libraries is increased by about ten times. Comparing with “3dRNA_Oldlib”, the prediction accuracy of “3dRNA_Lib1” and “3dRNA_Lib2” are improved considerably, not only for small molecules but also for RNAs with complicated or large structures.In order to ensure the synchronization of the template library of 3dRNA with newly added RNAs in PDB, we will automatically monitor the PDB database regularly in the later to add the SSEs of new RNAs into the template library. In future, we will also intend to enlarge the number of open loops to improve the prediction accuracy of 3dRNA since the missing of open-loop templates affects the prediction accuracy of RNA too.Availability and Implementationhttp://biophy.hust.edu.cn/new/3dRNA and the validation data can also be downloaded at the web server.The web server of 3dRNA with the new template library is available at Yi Zhang: Methodology, Investigation, Formal analysis, Visualization, Software, Validation, Writing - original draft. Jun Wang: Methodology, Software, Validation. Yi Xiao: Conceptualization, Supervision, Methodology, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


It has a wide non‐trivial band gap of 210 meV, which is generated by strong spin‐orbit coupling, and possesses protected electronic edge‐states. In the structure, charged layers of ∞2[(Bi4Rh)3I]2+ honeycombs and ∞1[Bi2I8]2− chains alternate. The non‐trivial topology of Bi14Rh3I9 is an inherent property of the 2D intermetallic fragment. Here, the exfoliation of Bi14Rh3I9 was performed using two different chemical approaches: (a) through a reaction with n‐butyllithium and poly(vinylpyrrolidone), (b) through a reaction with betaine in dimethylformamide at 55 °C. The former yielded few‐layer sheets of the new compound Bi12Rh3I, while the latter led to crystalline sheets of Bi14Rh3I9 with a thickness down to 5 nm and edge‐lengths up to several ten microns. X‐ray diffraction and electron microscopy proved that the structure of Bi14Rh3I9 remained intact. Thus, it was assumed that the particles are still TIs. Dispersions of these flakes now allow for next steps towards the envisioned applications in nanoelectronics, such as the study of quantum coherence in deposited films, the combination with superconducting particles or films for the generation of Majorana fermions, or studies on their behavior under the influence of magnetic or electric fields or in contact with various materials occurring in devices. The method presented generally allows to exfoliate layers with high specific charges and thus the use of layered starting materials beyond van der Waals crystals.The layered salt Bi Flaky insulation: Topological insulators are a novel class of quantum materials that have high application potential in quantum computers and spintronics. This work presents a scalable chemical protocol to exfoliate crystals of the topological insulator Bi14Rh3I9 into unsupported few layer thick flakes without decomposition. The new method is generally suitable to overcome strong electrostatic interactions in layered salts. These high‐melting metal carbides or nitrides have layered structures, but no van der Waals gaps. However, by etching with hydrofluoric acid the A component, in this case Al, can be removed topochemically and OH− or F− terminate the remaining transition metal carbide layers. In this way van der Waals gaps are generated, which allow the subsequent delamination of free‐standing metallic layers, named MXenes.Two‐dimensional (2D) materials have attracted significant attention due to their physical properties that distinctly differ from bulk materials.1, 2 ISuch 2D metals are of special interest, since quantum confinement leads to a density of state that markedly differs from bulk metals. As a consequence, the electronic resistance of 2D metals is independent of the path length. In contrast to the variety of semiconducting 2D materials, freestanding layers of metallic conductors are rare. The major reason for this is the scarcity of metallic bulk precursors with coherent networks of strong covalent bonds in only two dimension that are, for instance, separated by weak van der Waals gaps. In addition, monolayers of metal atoms are mostly only stable on substrate surfaces or with surfactantsMoreover, there is a contentious issue whether “ideal” 2D metals can exist at all. Anderson and Mott showed that any disorder or strong Coulomb interactions between electrons could cause a 2D metal to become an insulator. From this perspective any approach towards a 2D metal should aim at minimizing disorder as well as repulsive interactions between electrons, in particular the conduction electrons.20, 21In this context, the topological insulators (TIs) could be of utmost importance.22, 233 into 10 nm thick platelets will theoretically provide 100 m2 of 2D material. Moreover, not all of the relevant materials can be deposited from the gas phase. Possible starting materials are weak 3D TIs that comprise layered structures with strong in‐plane bonds and weak interactions between layers.Currently, high‐quality thin layers of TIs, which are desirable for real industrial applications, are made for example by atomic force microscopy,14Rh3I9, which has a wide topological band gap of about 210 meV.∞2[(Bi4Rh)3I]2+ alternate with layers formed by iodobismuthate(III) chains ∞1[Bi2I8]2− .14Rh3I9 as a suitable starting material for a liquid‐phase exfoliation. Quantum‐chemical calculations had shown that strong covalent Rh−Bi bonds together with weaker Bi−Bi bonds stabilize the intermetallic honeycomb net chemically as well as mechanically. The interaction with the anionic spacer is weaker and predominantly electrostatic. honeycomb changed its charge from +3 to +4 but remained fully intact, despite the harsh conditions, the considerable volume change, and the mass transport. Mechanical and chemical robustness of the layer as well as its ability to tolerate moderate bending are essential prerequisites for a delamination and exfoliation process. A major issue, however, is the high layer charge, which must be compensated. Since the most top‐down exfoliation methods are based on mechanical treatments and elaborated for layered materials that only bear van der Waals interactions, we developed two new exfoliation routes for layered salts such as Bi14Rh3I9.In 2016, Rasche et al. presented the weak 3D‐TI Bi14Rh3I9 is composed of charged layers and chains, there is an unconditional need for a charge compensation during the exfoliation. One option to consider is a reduction reaction that diminishes or eliminates the positive charge of the ∞2[(Bi4Rh)3I]2+ TI layers, thereby weakening the interaction between the layers. Either anionic ligands with good donor properties or surfactants may be used to stabilize the partially or uncharged intermetallic layers in order to avoid re‐aggregation. These charge‐neutral products may then be cleaved by mechanical activation like sonication, analogue to earlier reported methods used for neutral layered materials to produce freestanding, that is, not substrate‐supported, flakes. However, it is important to note that reduction to an uncharged ∞2[(Bi4Rh)3I]±0 layer involves the uptake of two electrons per formula unit. This change of the electron count shifts the Fermi level to another, fortunately also non‐trivial band gap of about 100 meV.Since Bi13Pt3I7.14Rh3I9 crystals were suspended in n‐hexane before the reducing agent nBuLi was injected under inert conditions. During that reductive treatment, the crystallinity and morphology of the starting material changed dramatically. The measured powder X‐ray diffraction (PXRD) pattern could no longer be assigned to the theoretical diffraction pattern of Bi14Rh3I9, but to rt‐Bi2Rh, rt‐Bi3Rh, and BiI3 (Supporting Information 1). Apparently, Bi14Rh3I9 decomposed.The first approach represents a heterogeneous reduction process based on the published topochemical approach applied on Bi14Rh3I9 crystals were reacted in n‐hexane and nBuLi together with a surfactant like poly(vinylpyrrolidone) (PVP). A tendency towards a separation of the dense parent crystal and a puff pastry like morphology were observed  or Bi12Rh3I. Local energy‐dispersive X‐ray spectroscopy (EDX) measurements indicated a ratio of Bi/Rh/I=77(3):20(3):3(2) at % . The mass transport and volume change led to porosity of the solid product and a shift of the remaining intermetallic layers into a primitive stacking sequence. The observed intensities and positions of the reflections in the electron diffraction experiment match very well the simulated electron diffraction pattern for an eclipsed layer stack . Although the betaine molecule has a positively and a negatively charged end, it is not a classical zwitterion, because it possesses no hydrogen atom, which can migrate and enable isomerization to a form without charge separation.14Rh3I9, but large enough to promote splitting by an additional solvent or even separation of the layered compound. Moreover, the anionic carboxyl group can be protonated and thereby the betaine molecule will be charged. After testing several solvents and reaction parameters, the most promising results were observed when treating Bi14Rh3I9 with betaine in dimethylformamide (DMF) in the temperature range between 55 and 60 °C for at least 24 h.In the first step of the substitutive approach, we aimed at substituting the anionic spacer of Bi14Rh3I9 crystals did not change as dramatically as in the reductive approach. The crystals swelled along the stacking direction and fanned out like a stack of cards, with each card  straight and well separated from its neighbors . The same applied to a reaction in which Bi14Rh3I9 was heated in betaine together with another solvent, for example, ethanol or acetone (Supporting Information 3).A higher reaction temperature fostered the expansion of the layered compound, at least to the point at which decomposition and collapse of the expanded stack took place. A higher concentration of betaine gave a higher yield of exfoliated thin layer packages, but only up to the solubility limit of betaine in DMF. The maximum of crystal splitting and the highest yield of separated thin flakes was obtained at 55 °C after 72 h. In experiments in which Bi14Rh3I9, though the mechanism is not known up to now. UV/Vis spectra hint at the formation of an intermediate upon heat treatment of the mixture (Supporting Information 4). Commercially available DMF absorbs at 210 and 244 nm, an aqueous solution of betaine at 210 nm. In contrast, the spectrum of a solution of betaine in DMF that was heated to 60 °C in analogy to the exfoliation conditions shows two additional signals at 252 and 265 nm. PXRD (Supporting Information 5) and EDX analysis confirmed that the chemical composition of the flakes (Bi/Rh/I=55(2):13(3):31(3) at %) agrees to the chemical composition of the starting material Bi14Rh3I9 (54:12:34 at %). The preservation of the entire crystal structure was supported by SCXRD  studies on selected flakes (no sonication) showed a thickness between 200 and 800 nm and a lateral dimension up to several ten microns . Subsequently, the dispersion was separated in particle size dependent fractions by (ultra)‐centrifugation. The few‐layer sheets were characterized via TEM analyses TEM images of distinct areas show an ordered sequence of a hexagonal motif, which fits well with the intermetallic layers of Bi14Rh3I9. EDX analyses of flakes revealed a composition of Bi/Rh/I=66(6):7(2):28(4) at %. These findings strongly suggest that we succeeded in exfoliating thin flakes of the weak 3D topological insulator Bi14Rh3I9.To enhance swelling and layer separation, a tip sonication treatment was performed (The thickness of the flakes was determined by off‐axis electron holography (EH) studies.44, 4514Rh3I9 crystals . were collected using an X′Pert Pro diffractometer . The samples were ground and fixed on single‐crystal silicon sample holders. The Powder Diffraction File Database (PDF) and the Inorganic Crystal Structure Database (ICSD) were used for phase identification.Single‐crystal X‐ray diffraction: Single‐crystal X‐ray diffraction measurements  were performed on a four‐circle diffractometer Kappa APEX II CCD (Bruker) equipped with a graphite(002) monochromator, and a CCD detector. Single‐crystals were selected and glued on top of a glass fiber, which was then mounted to a goniometer head. Some of the crystals were washed with ethanol or cut in inert oil in order to provide suitable quality and size for the measurement. Polarization and Lorentz factors corrections were performed within the APEX2 program.Scanning electron microscopy and energy‐dispersive X‐ray spectroscopy: SEM was performed using an electron microscope SU8020 from Hitachi equipped with multi detector system for secondary and low‐energy backscattered electrons while an Oxford Silicon Drift Detector X‐MaxN was used for the semi‐quantitative EDX. Suspensions washed with ethanol were dropped on a silicon wafer fixed on a carbon pad under argon, which were then settled on an aluminum sample holder. The EDX data were evaluated within the AZtec software package.Transmission electron microscopy and off‐axis electron holography: TEM imaging was performed in an FEI Titan3 80–300 (ThermoFisher Company) microscope at 300 kV acceleration voltage. The instrument was equipped with an image aberration corrector allowing for a spatial resolution down to 0.7 Å in the bright field mode. We conducted both high‐resolution TEM imaging and electron diffraction studies to resolve the crystal structure. To record the diffraction patterns we therefore used the three‐lens condenser system to generate a parallel illumination at larger probe diameters of about 400 nm, significantly reducing the beam induced damage. To index the diffraction patterns we employed the SingleCrystal software package  allowing for simulating electron diffraction patterns.3 80–300 using an acceleration voltage of 300 kV. To image larger regions of the few 100 nm sized flakes, the holographic field‐of‐view was enhanced by using the so‐called Lorentz mode . In order to generate an electron hologram, that is, the interference pattern between the object‐exit wave and object‐free reference wave, a bias voltage of 180 V was applied to the electro‐static Moellenstedt biprism. Phase images of the Bi14Rh3I9 object exit waves were then reconstructed from the holograms by Fourier methodφ and thickness t, according to φ=Cϵ⋅V0⋅t, the thickness of the flakes was determined with known interaction constant Cϵ=0.0065 rad V−1 nm−1 at 300 kV and mean inner potential V0=16.3 V of Bi14Rh3I9.Off‐axis electron holographyAtomic force microscopy: AFM measurements were taken with a Dimension Icon (Fa. Bruker) with a cantilever. The images were plotted with the NanoScope 9.3 Software. Therefore, samples were prepared by spin‐coating on poly(dimethylsiloxane) (PDMS) stripes, which were fixed in a provided module. The PDMS surface was shortly heated using a hotplate to evaporate solvent residues.UV/Vis spectroscopy: UV/Vis absorption spectra were measured with a UV‐1650PC spectrometer (Shimadzu) using quartz glass SUPRASIL cuvettes . The measurements were taken in the wavenumber range from 200 nm to 800 nm.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Diabetes aggravates the risk and severity of periodontitis, but the specific mechanism remains confused. Complement 3 (C3) is closely related to complications of type 2 diabetes (T2DM). In the present study, we concentrated on whether C3 mediates the development of periodontitis in T2DM.Levels of C3 in blood and gingival crevicular fluid (GCF) of patients were measured first. A C3‐knockout diabetic mouse model was established, real‐time PCR, Western blotting and histological investigation were performed to evaluate the progress of periodontitis. Microcomputed tomography (micro‐CT) and TRAP staining were performed to detect alveolar bone resorption. Immunofluorescence was performed to detect polarization of macrophages.Our data showed that C3 levels were elevated in the blood and GCF of T2DM patients compared with non‐diabetic individuals. Increased C3 was closely related to the upregulation of inflammatory cytokines including interleukin (IL)‐1, IL‐6 and tumour necrosis factor‐alpha (TNF‐α), as well as the decline of the bone volume density (BMD) and bone volume over total volume (BV/TV) of the alveolar bones in diabetic mice. The deletion of C3 inhibited inflammatory cytokines and rescued the decreased BMD and BV/TV of the alveolar bones. C3‐mediated polarization of macrophages was responsible for the damage.T2DM‐related upregulation of C3 contributes to the development of periodontitis by promoting macrophages M1 polarization and inhibiting M2 polarization, triggering a pro‐inflammatory effect on periodontal tissues. T2DM led to an increase in C3 levels, which affected the macrophages in periodontal tissues by promoting macrophages to polarize to M1 and inhibiting M2 polarization, thus triggering a pro‐inflammatory effect on periodontal tissues. C3‐related osteoclasts increase potentially promoted alveolar bone resorption. In addition, preliminary mechanism was explored. In the present study, the levels of C3 in blood and gingival crevicular fluid (GCF) of patients were measured first, and a C3‐knockout diabetic mouse model was established to study the roles of C3 in the progression of T2DM‐related periodontitis.22.1A total of 17 T2DM patients in the Department of Endocrinology, the Second Affiliated Hospital of Xi'an Jiaotong University, Shaanxi Province, China were included in this study. T2DM was diagnosed according to the standard of the American Diabetes Association published in 2018.The inclusion criteria of T2DM: patients diagnosed with T2DM for more than six months and in stable condition; good periodontal conditions without gingivitis or periodontitis; no antibiotic or immunosuppressant treatment in the past three months; not pregnant; and no acute infections or allergies. The inclusion criteria of the non‐diabetic patients: individuals with normal blood glucose without type‐1 diabetes or T2DM; who had good periodontal conditions without gingivitis or periodontitis; who had no systemic diseases; who did not consume antibiotics or immunosuppressants in the past three months; who were not pregnant; and who had no acute infection or allergy. The inclusion criteria of periodontitis: patients diagnosed with periodontitis without any periodontal treatments in the last 6 months; who with normal blood glucose without type‐1 diabetes or T2DM; no antibiotic or immunosuppressant treatment in the past three months; not pregnant; and no acute infections or allergies. The inclusion criteria of diabetic periodontitis: patients diagnosed with T2DM for more than six months and in stable condition; patients diagnosed with periodontitis without any periodontal treatments in the last 6 months; no antibiotic or immunosuppressant treatment in the past three months; not pregnant; and no acute infections or allergies.All included patients signed the written informed consent and agreed to the test of C3 content in blood and GCF and the routine periodontal examinations. All operations were performed in accordance with the relevant ethical regulations of Xi'an Jiaotong University and passed the ethical review of the Secondary Affiliated Hospital of Xi'an Jiaotong University and the Stomatological Hospital (College) of Xi'an Jiaotong University.2.2Six millilitre of peripheral blood was collected from all patients with empty stomach in the morning, serum was centrifuged at 4°C, 3000 r/min, 15 minutes, and then stored at −70°C. Filter paper (Whatman) was prepared into 2 mm × 8 mm size. Bilateral maxillary and mandible molars (The third molars were not included) were tested at mesial, distal and buccal positions. For GCF collection, the filter paper was inserted into the sulcus for 30 seconds and washed by centrifugation with 1% PBS. Bleeding on Probing (BOP), gingival index (GI), probing depth (PD) and tooth mobility (Mob) were evaluated for each patient.2.3C57BL/6 male mouse weighing ~23 g (n = 40) was used in the present study. All mice were housed under standard conditions with a 12 hour light/dark cycle in a specific‐pathogen‐free (SPF) facility. Mice were randomly divided into four groups. The control group was fed with basic food for 4 weeks before 4 weeks of injection with normal saline (n = 10). The STZ group was fed with basic food for 4 weeks before 4 weeks of injection with streptozotocin  (n = 10). Four weeks of high‐sugar and high‐fat food followed by 4 weeks of normal saline injection were performed as HF/HS diet group (n = 10). Four weeks of high‐sugar and high‐fat food followed by 4 weeks of low dose (60 mg/kg) STZ intraperitoneal injection were performed to simulate T2DM (n = 10). Fasting glucose was detected using a glucose analyzer, and fasting glucose ≥ 16.7 mmol/L was determined as diabetic status.2.4® Premix Ex Taq™ II (TaKaRa). Sequences of the primers are provided in Table Gingiva was harvested from the maxillary second molars and stored at −70°C for real‐time PCR. Total RNA was isolated using the Trizol reagent (Invitrogen) according to the manufacturer's instructions and diluted in 30 μL of RNase‐free water. Equal amounts of total RNA (2 μg) were reverse transcribed with a First Strand cDNA Synthesis Kit (Fermentas). Gene expression was analysed using an iQ5 (Bio‐Rad) with SYBR2.5Rabbit Anti‐C3 antibody  and β‐actin  antibody (Abcam) were used. Protocols of Western blotting were used as previously described.2.6Maxillary bones were excised and immediately fixed in 4% paraformaldehyde neutral buffer solution for 48 hours. Then, the maxillary specimens were decalcified with 10% EDTA at room temperature for 7 days until the alveolar bone could be easily penetrated followed by conventional dehydration and paraffin embedding. Immunohistochemical detection of C3 and RANKL as well as HE staining was performed in each group of samples. Examination and analysis were performed in blind. For immunohistochemical detection, 5 μm sections were prepared. Deparaffinized sections were treated with methanol containing 3% hydrogen peroxide before conducting antigen retrieval using a microwave oven at 95°C for 5 minutes, and cooling at room temperature for 15 minutes for two times. After washing with phosphate‐buffered saline (PBS), 5% bovine serum albumin was applied for 10 minutes. The sections were incubated with antibody 16‐18 hours overnight at 4°C. Then, after washing by PBS, a biotin‐conjugated secondary antibody was applied for 30 minutes. DAB chromogenic agent kit (Boster) was used to develop colour and the samples were counterstained with haematoxylin. Antibodies used were Rabbit Anti‐C3 antibody  and Rabbit Anti‐RANKL antibody .2.7Bilateral maxillary was fixed in 4% paraformaldehyde. Tissues were scanned on a micro‐CT system. The parameters are set as follows: 70 kV, 114 μA, 12 μm resolution; Root bifurcation area of second molar was defined as the volume of interest (VOI). Thirty slices prior to and after the identified furcation slice were added to generate a VOI. The distance between cemento‐enamel junction and the alveolar bone crest (CEJ‐ABC) was assessed. Bone volume density (BMD) and bone volume over total volume (BV/TV) were analysed.2.8TRAP staining was performed by using Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma) according to the manufacturer's instructions. Briefly, slices were deparaffinized and rinsed with deionized water. TRAP staining was performed at 37°C in the dark for 1 hour. Nuclei were stained with haematoxylin for 2 minutes.2.9Detailed methods are described as previously.2.10t test were performed to analyse differences among or between groups using SPSS 16.0 software . In all analyses, a P value < .05 was taken as the level of significance.The quantitative data shown in the figures are presented as the mean ± SD. One‐way analysis of variance (ANOVA) and Student's 33.1In this study, we compared the levels of C3 and its fragments in the blood of non‐diabetic individuals, T2DM patients, periodontitis patients and diabetic periodontitis patients. Table 3.2The weight of GCF was gained in the T2DM group compared with non‐diabetic individuals, while the weight gained even more in the periodontitis patients and diabetic periodontitis patients Figure . The lev3.3According to Figures 3.4To further prove that C3 is a key molecule for T2DM to promote periodontal disease, we established a C3 knockout mouse model and induced them with T2DM. The knockout effect of the mouse model was first verified in the gingival tissues by Western blotting and immunohistochemistry, showing that C3 expression was strongly inhibited Figure . Decreas3.5In this study, we also assessed osteoclasts in the alveolar bone and showed that they increased significantly in the diabetic mice, while their number in alveolar bone decreased after the deletion of C3 Figure . RANKL wWe next investigated the mechanism of C3 regulation in the development of T2DM‐associated periodontal destruction. C3 was considered to be closely related to macrophages. The expression of C3 can affect the polarization of macrophages, promoting M1 polarization while inhibiting M2 polarization. Macrophage polarization has dual role as killers (M1) or builders (M2) in chronic inflammatory diseases, such as periodontitis. Therefore, we next examined the polarization of macrophages in the periodontal tissues. As shown in Figure 4In this study, we found that the increase in C3 levels of T2DM patients may be the cause of periodontal destruction. Although it did not cause obvious bone destruction, the increase in the number of osteoclasts may aggravate periodontal bone destruction. Increase in C3 caused macrophages in the periodontal tissues to polarize to M1 and inhibited the M2 polarization which may be a mechanism by which it promotes periodontitis.Diabetes affected bacteria‐host interactions to promote inflammation and periodontal disease.In the present study, we first fed mice with high fat and high sugar food for 4 weeks to induce insulin resistance, then low dose of STZ injection was followed for next 4 weeks to destruct islet tissues to establish T2DM model. Basic diet with STZ injection (T1DM) did not change the expression of C3, which was in line with previous studies.C3 can be cleaved into C3a and C3b by C3 convertase.Periodontal inflammation is a chronic and long‐term process. Although no bone destruction was found over the short‐term, micro‐CT showed that BMD and BV/TV decreased in the diabetic mice. Trap staining showed a significant increase in the number of osteoclasts, and damage to the alveolar bone possibly appeared at subsequent time points. Since some serious complications were observed in diabetic mice, we did not extend the observation period due to ethical considerations. Deletion of C3 rescued the periodontal inflammation phenotype in diabetic mice, suggesting that the control of C3 levels in diabetic patients may reduce the risk of periodontal disease at an early stage. We found that an increase in C3 caused upregulation of the inflammatory cytokines IL‐1, IL‐6 and TNF‐α in the gingiva, inducing M1 macrophage polarization and inhibiting M2 polarization. Macrophages are very important immune cells and are closely related to the complement system.5In summary, this study showed that C3 levels were significantly increased in the blood and GCF of T2DM patients. Increased C3 levels were accompanied by periodontal destruction. Deletion of C3 extenuated gingival inflammation and rescued potential bone destruction. C3 increased M1 macrophage polarization and decreased M2 polarization, suggesting that C3 may promote the development and progression of periodontal inflammation by regulating macrophages polarization.The authors declare no conflicts of interest to the authorship and/or publication of this article.Ye Li, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript. Xinxin Wang, contributed to conception, design, data analysis and interpretation, critically revised the manuscript. Saisai Wang, Chunhui Zhu and Jing Guo contributed to data acquisition, analysis, and interpretation, critically revised the manuscript. Ke Li, contributed to conception, data acquisition and analysis, critically revised the manuscript. Ang Li, contributed to conception, design, data analysis and interpretation, drafted and critically revised the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the present work.All experiments were reviewed and approved by the Ethics Committee of College of Stomatology, Xi'an Jiaotong University.


Arabidopsis thaliana, a floral repressor, AtFLC, undergoes epigenetic silencing. Although the silencing of AtFLC is maintained under warm conditions after a sufficient duration of cold, FLC orthologues are reactivated under the same conditions in perennial plants, such as A. halleri. In contrast to the abundant knowledge on cold requirements in AtFLC silencing, it has remained unknown how cold duration affects the reactivation of perennial FLC. Here, we analysed the dynamics of A. halleri FLC (AhgFLC) mRNA, H3K4me3, and H3K27me3 over 8 weeks and 14 weeks cold followed by warm conditions. We showed that the minimum levels of AhgFLC mRNA and H3K4me3 were similar between 8 and 14 weeks vernalisation; however, the maximum level of H3K27me3 was higher after 14 weeks than after 8 weeks vernalisation. Combined with mathematical modelling, we showed that H3K27me3 prevents a rapid increase in AhgFLC expression in response to warm temperatures after vernalisation, which controls AhgFT expression and the initiation of flowering. Thus, the duration of cold defines the rate of AhgFLC reactivation via the buffering function of H3K27me3 against temperature increase.Vernalisation is the process in which long-term cold exposure makes plants competent to flower. In vernalisation of  Studies investigating the natural variation in vernalisation requirement have identified one of the main responsible genes, FLOWERING LOCUS C (FLC) in Arabidopsis thaliana (hereafter AtFLC)4. Molecular genetic studies have revealed that AtFLC, which encodes a MADS-box protein, represses flowering before vernalisation in winter annual ecotypes6. AtFLC suppresses flowering, at least in part, by repressing the expression of the floral activators AtFT, AtFD, and AtSOC18.Vernalisation, a major flowering pathway, is the process that makes plants competent to flower following long-term cold exposureA. thaliana, AtFLC undergoes quantitative silencing, which depends on the duration of cold9. This process is accompanied by the decrease in active histone modifications, especially histone H3 lysine 4 trimethylation (H3K4me3), at a region around the transcription start site known as the nucleation region, and the increase of a repressive histone modification, histone H3 lysine 27 trimethylation (H3K27me3), at the same region16. After return to warm conditions, H3K27me3 spreads to the entire gene locus and silencing is maintained16, which induces the expression of floral activators and flowering under favourable long-day conditions9. The duration of cold required for full vernalisation is different between A. thaliana accessions19. Accessions from Sweden require a longer duration of cold for stable AtFLC repression compared with accessions from other countries, and variation in the vernalisation response is correlated with epigenetic silencing of AtFLC19. In Col FRISf2, the H3K27me3 level at the nucleation region of AtFLC was found to be saturated after 6 weeks of cold15. In Lov-1, which was collected from northern Sweden, H3K27me3 accumulation at AtFLC was delayed and a longer duration of cold was required to reach H3K27me3 levels equivalent to those in Col FRISf219.In the vernalisation process of AtFLC silencing process, the effect of cold duration on the upregulation of perennial FLC orthologues on return to warm has remained to be determined. FLC orthologues are silenced in a quantitative manner under cold conditions as well as A. thaliana; however, they are upregulated under warm conditions following vernalisation in perennial plants, Arabidopsis halleri25, Arabidopsis lyrata26, and Arabis alpina29. H3K27me3 accumulates at perennial FLC orthologues under cold conditions and is removed from the locus in response to warm temperatures27. In perennial plants, the upregulation of FLC orthologues under warm conditions following vernalisation would antagonise the upregulation of FT orthologues and flowering. Thus, it is expected that an insufficient cold duration would affect flowering through the perturbation of perennial FLC upregulation.In contrast to the abundant knowledge on cold requirements in the 33. We previously reported the seasonal dynamics of AhgFLC mRNA, H3K4me3, and H3K27me3 over 2 years in a natural population, and constructed mathematical models to analyse the properties of the regulatory system24. Numerical simulations revealed that bidirectional interactions between H3K27me3 and H3K4me3 are required for AhgFLC expression to respond to long-term temperature trends in a fluctuating natural environment24.A mixed approach, including time series data and mathematical modelling, has been used to investigate the molecular mechanisms underlying the vernalisation processAhgFLC mRNA, H3K4me3, and H3K27me3 over 8 weeks and 14 weeks of cold conditions followed by warm conditions , and then performed numerical simulations to determine the role of H3K27me3 in the AhgFLC regulation over the two conditions. We showed that the minimum levels of AhgFLC mRNA and H3K4me3 were similar between the 8 weeks and 14 weeks vernalisation treatments, while the maximum level of H3K27me3 was higher in the 14 weeks than in the 8 weeks vernalisation treatment. Combined with mathematical modelling, we showed that H3K27me3 plays a key role in moderating the rate of AhgFLC reactivation and controls the initiation of flowering.In this study, we analysed the dynamics of AhgFLC mRNA levels, and the H3K4me3 and H3K27me3 levels at six amplicons along the locus using a chromatin immunoprecipitation (ChIP), in the 8 weeks and 14 weeks vernalisation treatments  which was established to predict the mRNA and histone modification dynamics in a natural population of A. halleri24 and the model that lacks H3K27me3 (model 2). In model 1, H3K27me3 and H3K4me3 were assumed to antagonise each other at the two nucleation regions of AhgFLC, which was represented by differential equations. The mRNA level was modelled by linear regression with the H3K4me3 level at the nucleation region. The assumptions in model 2 is the same as those in model 1 except that all variables and parameters regarding the H3K27me3 states were excluded from the full model (see the Methods section for more detail). We optimised the parameters in the two models to fit the model outputs to the observed data. In model 1, the simulated H3K4me3 and H3K27me3 levels at the nucleation region—which are directly linked to the mRNA level—were highly correlated with the observed data  around TSS of Lov-1 AhgFLC expression in a natural fluctuating environment24. In this study, we analysed the responses to cold and warm temperatures separately, and showed that H3K27me3 prevents a rapid reactivation of AhgFLC although H3K27me3 is finally reset in response to warm conditions after vernalisation. This indicates that H3K27me3 may function as a buffer against a rapid temperature increase, moderating the change speed of AhgFLC rather than maintaining the silenced state. Consequently, AhgFT expression is induced within a time window when AhgFLC expression is low, leading to flowering. In addition, our data revealed that the duration of cold exposure could explain the lower level of H3K27me3 at AhgFLC reported in laboratory samples (after 6 weeks cold) compared with field samples (in winter)34.Previously, we reported that H3K27me3 is required for the long-term responses of AhgFLC expression is higher at warmer temperatures, and predicted that the flowering period decreases with climate warming based on a mathematical modelling21. Consistently, we showed that early AhgFLC reactivation due to an inadequate period of cold delays the initiation of flowering. These studies focused on the time to flower, and the influence of high AhgFLC expression on plant fitness should be addressed: we need to measure other reproductive traits, such as total flower numbers in future studies. The time-series analyses of reactivation of perennial FLC performed in this study will help reveal the molecular basis of reproductive strategies of plants with various life histories.A previous study showed that A. halleri plants growing in a natural population at Monzen, Naka-ku, Taka-cho, Hyogo Prefecture, Japan . Plants were selected along a stream in a sampling area of approximately 20 m × 230 m. The area was divided into three sections, and eight plants were selected from each section (> 2 m between plants). Four plants from each section were allocated to each of the 8 weeks and 14 weeks vernalisation treatments . The plants were transferred from the field to the laboratory on 29 October 2013, when AhgFLC was highly expressed24. The plants were transplanted into pots and grown on outdoor pot shelves at the Center for Ecological Research, Kyoto University, Otsu, Shiga prefecture, Japan . After a 10-day period of growth adjustment in the outdoor pots, the plants were grown under cold conditions at 4 °C with 12 h light/12 h dark for 8 or 14 weeks, and then grown under warm conditions at 20/15 °C with 12 h light/12 h dark. We used the same photoperiod between cold and warm conditions to measure the effect of temperature on plant phenology separately from photoperiod. The 12 h light/12 h dark condition imitates the day length in our field site in spring (March–April) when plants start reproductive growth24. The maximum and minimum day length in our field site are approximately 14 and 10 h, respectively20.For the vernalisation experiment, we selected 24 later Stabilization Solution  on ice during sampling and stored at − 20 °C until RNA extraction. For ChIP samples, one fully expanded young leaf (approximately 0.1 g) was harvested from each of the 12 plants, and four biological replicates were prepared by pooling three leaves per replicate (0.3 g per replicate) on each sampling date. Harvested leaves were fixed with 1% formaldehyde in phosphate-buffered saline (PBS), using vacuum infiltration twice for 5 min each. The cross-linking reaction was quenched with the addition of glycine to a final concentration of 125 mM and vacuum infiltration once for 5 min. Cross-linked samples were washed with PBS, frozen in liquid nitrogen, and stored at − 80 °C until chromatin extraction.Leaves were sampled at 13–17 days interval under cold conditions, and 3–7 days interval after transfer to warm conditions. On each date, sampling started at 12.00, and was completed within 1.5 h (by 13.30). For RNA samples, one small young leaf (approximately 0.01 g) was harvested from each of the 12 plants, and four biological replicates were prepared by pooling three leaves for each replicate on each sampling date. Harvested leaves were preserved in RNAAhgFLC mRNA levels were normalised to those of either AhgACT2 or AhgPP2AA334.RNA was extracted using an RNeasy Plant Mini Kit . The extracted RNA was quantified using a Qubit Fluorometer and Qubit RNA HS Assay Kits (Thermo Fisher Scientific). cDNA was synthesised using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). qPCR was performed in duplicate with the appropriate primers  at 10% power output. Samples were centrifuged, and the supernatant was diluted in ChIP dilution buffer (up to 900 μl). This solution was incubated with Dynabeads Protein G (Thermo Fisher Scientific) at 4 °C for 1 h with rotation for pre-clearing. The solution was then incubated with antibodies for 5 h. The antibody dilutions were as follows: 1:500 for anti-H3K27me3  and anti-H3K4me3 , and 1:1,000 for anti-histone H3 . After incubation with the antibody, Dynabeads Protein G was added, and samples were incubated at 4 °C for an additional 2 h. After immunoprecipitation, the beads were washed sequentially with low salt, high salt, LiCl, and TE buffers. The chromatin was then eluted with elution buffer, cross-linking was reversed by heating at 65 °C for 12 h, and proteinase K treatment was conducted at 45 °C for 1 h. DNA was extracted using phenol/chloroform, ethanol-precipitated, and then resuspended in 50 μl of TE buffer. qPCR was performed in duplicate by using the appropriate primers , in both the 8 weeks and 14 weeks vernalisation treatments. Bolting was defined as the stage when flowering stems were longer than 3 mm before presenting open flowers. Flowering was defined as the stage when the flowers had opened. Reversion was defined as the stage when leaves had formed at the top of flowering stems at the end of the flowering period. During reversion, plants form aerial rosettes and generate roots at the top of flowering stems and most of the lateral meristems (nodes of cauline leaves), facilitating the establishment of vegetative  offspring.24 was used. In this model, H3K27me3 at the AhgFLC locus was classified into four states: UNUD (neither nucleation regions are modified), MNUD (only the nucleation region is modified), MNMD (both nucleation regions are modified), and UNMD —the proportions: uNuD, mNuD, mNmD, and uNmD, respectively. The transition of H3K27me3 at the locus was assumed to be unidirectional, that is, UNUD → MNUD → MNMD → UNMD → UNUD. The H3K4me3 states were described by UN and AN for the nucleation region (the proportions: uN and aN), and UD and AD for the distal nucleation region (the proportions: uD and aD).For the full model (model 1 with H3K27me3), the mathematical model established by Nishio et al.NUD → MNUD and MNUD → MNMD were assumed to be induced by cold and warm temperatures, represented by the functions of temperature, μ(T) and ν(T), respectively. These functions are given by the logistic equations of the form:NMD → UNMD transition was assumed to be induced by H3K4me3 at the nucleation region (aN), and the UNMD → UNUD transition was assumed to occur at a constant rate (λ). Thus, the time derivatives of the H3K27me3 states at the AhgFLC locus are given byξ(T)] and cold [τ(T)] temperatures, respectively. These functions are given by the logistic equations of the form:AhgFLC locus are given byThe transitions UmNuD + mNmD and mNmD + uNmD were multiplied by the maximum observed levels of H3K27me3 at the nucleation region and the distal nucleation region, respectively; aN and aD were multiplied by those of H3K4me3 at these regions, respectively.The observed histone modification levels at the nucleation region and the distal nucleation region were represented by the mean level of amplicons I and II, and the level of amplicon VI, respectively. To represent the simulated histone modification levels in each vernalisation treatment, 38 using the optim function of R. Explanations and the determined values of the parameters are listed in Supplementary Table Simulations were performed using the ode function of the deSolve package of R v3.3.2. Model parameters were optimised by minimising the residual sum of squares between the model output and the observed data in both vernalisation treatments in two steps. First, we randomly selected 1000 parameter sets from the uniform distribution, and selected the parameter set that best fitted the observed data. Using the selected parameter set as the initial values, the parameters were then optimised by the simulated annealing (SANN) methodAhgFLC mRNA level by linear regression with the observed H3K4me3 level at the nucleation region given byWe modelled the observed AhgFLC mRNA level from the simulated H3K4me3 level at the nucleation region. The simulated mRNA level was standardised to ensure that the amplitude was even between models.Using the determined regression coefficient and intercept, we reproduced the simulated AhgFLC locus were excluded from the model. The time derivatives of the H3K4me3 states at the AhgFLC locus are given byAhgFLC mRNA level from the simulated H3K4me3 level at the nucleation region.For the model that lacks H3K27me3 (model 2 without H3K27me3), all variables and parameters regarding the H3K27me3 states at the d by Eq. , we reprSupplementary file 1.