MCM7, a subunit of mini-chromosome maintenance proteins (MCM) complex, plays an important role in initiating DNA replication during the G1 phase and extending DNA strands during the S phase. Here, we demonstrated that MCM7 is not only sustained but maintains association with chromatin during M phase. Remarkably, MCM7 siRNA can accelerate mitotic exit. MCM7 depletion leads to CDK1 inactivation and promotes subsequent cohesin/RAD21 cleavage, which eventually leads to sister chromatin segregation. Moreover, MCM7 is co-localized with tubulin in the mitotic cells and MCM7 depletion results in aberrant mitosis. Our results indicate that MCM7 may exert certain functions on spindle formation to prevent cytokinesis during early mitosis by regulating CDK1 activity. Recent studies have determined that there are more MCMs bound onto chromatin than required by replication origins, and the phenomenon is coined as the “MCM paradox”. The mechanism of the paradox is still unknown. Recent cancer studies have also suggested that MCM2-7 complex overexpression is relative to proliferation and malignancy of various cancers12.Eukaryotic DNA replication is a methodical and strictly controlled process evolved to ensure accurate replication. Many mistakes that occur during DNA replication can cause genomic instability. The pre-replicative complex (Pre-RC), which plays an important role in initiating DNA replication, consists of the origin recognition complex (ORC), CDC6, CDT1 and mini-chromosome maintenance proteins (MCMs). MCM2-7 is a heterohexamer complex which contains six MCM homologues. MCMs are determined to be DNA helicases involved in the initiation of DNA replication. MCM subunits are evolutionarily conserved in organisms ranging from yeast to human including features such as the ATPase motifs13. MCM7 also interact with ATR-interacting protein (ATRIP)-ATR, which is activated in the presence DNA replication damage14. Furthermore, the MCM7 gene codes for an important microRNA cluster miR-106b-25, and contribute to the induction of megakaryocytes polyploidy15. In vitro, MCM7 is highly expressed in multiple malignancies. Immunohistochemical studies in a variety of tissues show increased expression of MCM7 in several solid tumors, including melanoma16, endometrial carcinoma17, esophageal adenocarcinoma18, oral squamous cell carcinoma19, colorectal adenocarcinoma20, glioblastoma21 and thyroid cancer22.MCM7, a key component of the MCM complex, forms a double trimeric complex with MCM4 and MCM6. The double trimeric complex initiate DNA replication through unwinding DNA double strand. During S phase, the complex participates in elongating DNA stands. However, a growing body of evidence has demonstrated that the function of MCM7 is not limited to starting DNA replication. Recent studies suggest that MCM7 interact with the polo-box domain (PBD) of endogenous PLK1, a kinase expressed during S, G2 and M phases of the cell cycle and plays an important role in dissociating the cohesion from chromosomeIn this study, mRNA copy number and expression of MCMs were examined during cell cycle progression. We have also explored the correlation between MCM7 depletion and the M phase process. From this we have discovered that MCM7 depletion accelerates the M phase by promoting CDK1 phosphorylation and cleaving Cohesin/RAD21, allowing sister chromatids to segregate.To identify the mRNA expression levels of MCMs in a single cell cycle, HepG2 cells and UMUC-3 cells were synchronized at G1 phase by double thymidine block. Cells were then incubated in fresh medium and collected at 0 h, 3 h, 6 h, 9 h, 12 h, and 15 h. HepG2 cells were successfully synchronized in G1 phase (G1: 86.34% ± 0.62% at 0 h) and slowly entered the S phase at 6 h. After incubation in fresh medium at 9 h, most cells progressed into the G2/M phase (G2/M: 60.30% ± 2.93%) and entered the next cell cycle by the 15 h timepoint. Compared to HepG2, UMUC-3 demonstrated quicker cell cycle progression. UMUC-3 were arrested in G1 (G1: 63.11% ± 2.13%) at 0 h. After incubation in fresh medium, at about 6 h, most cells had entered the G2/M phase (G2/M: 40.61% ± 3.87%) and entered the next cell cycle after 9 h Fig. .Figure 1To confirm the cell cycle progression of the above cells, the mitotic marker p-Histone (Thr3) was examined by Western Blotting. In HepG2 cells, p-histone H3 was detected after releasing 9 h and deceased after releasing 12 h, which was consistent with FCM result. Similar results were observed in UMUC-3 cells Fig. .To determine MCMs’ mRNA expression in one cell cycle, MCMs’ mRNA isolated in these time points were detected by QPCR Fig. . We founChromatin-binding assay were performed to identify distribution of MCMs corresponding to cell cycle progression. In HepG2 cells, chromatin-binding MCM5 were decreased gradually as cells progressed into the G2/M phase. However, large amount of MCM4, MCM6 and MCM7 remained in chromatin in G2/M cells. MCM7 differed from the other MCMs in that it maintained binding to chromatin through the cell cycle Fig. . In UMUCSubsequently, we performed the chromatin-binding assay in human lung carcinoma SPC-A1 cells. SPC-A1 cells were synchronized by double thymine block in G1 (G1: 45.23% ± 1.20%). Cells entered G2/M phase after incubation in fresh medium at 6 h (G2/M: 44.51%). Most cells exit M phase after incubation in fresh medium at 9 h Fig. . The preTo observe MCM7 expression directly in the cells, MCM7 was detected via immunohistochemistry (ICH). MCM7 has high levels of expression throughout the whole M phase, including prophase, metaphase, anaphase and telophase Fig. .Figure 4Based on the latter results, it is confirmed that MCM7 maintains presence and association with chromatin during G2/M phase. To examine its function in the G2/M phase, MCM7 was depleted by RNAi in nocodazole-blocked G2/M cells. After being treated with 0.2 µg/ml nocodazole for 24 h, most HepG2 were blocked in the G2/M phase (G2/M: 89.33% ± 2.01%). After transferring into fresh medium from 0 h to 4 h, cell gradually exited M phase. The RNAi transfection successfully reduced MCM7 expression Fig. . In the During M phase, CDK1 inactivation is the key event for activating separase for cleaving Cohesin/RAD21, allowing sister chromatids to segregate. Therefore, we monitored changes in RAD21 and p-Cdk1 levels (p-Cdk1 is a conserved tyrosine phosphorylation (Tyr15 in humans), leading to inhibition of Cdk1 activity). In the control group, after nocodazole release, the p-CDK1 levels were higher in the earlier time points  and the RAD21 was gradually cleaved during mitosis exiting. Interestingly, the MCM7 levels were gradually increased. In MCM7 siRNA group, the p-CDK1 levels were kept much higher levels than in the control group indicating that Cdk1 activity was inhibited. Correspondingly, the RAD21 levels were decreased significantly Fig. . These rTo further characterize MCM7 in mitosis, HepG2 cells were synchronized by nocodazole and then incubated in fresh medium for 0, 0.5, 1 and 2 h, immunofluorescence was performed to detect the localization of tubulin and MCM7. In the control, cells were retained in mitosis from 0 to 2 h. Compared to control group, cells exited from mitosis at 2 h after MCM7 depletion Fig. . Moreove23.In our study, we have found that MCM2-7 mRNA decreased during a cell cycle. More intriguingly, MCM2-7 mRNA was lowest in the G2/M phase. We also found that the reduction was not gradual. MCM2-7 mRNA increased in the S phase and then gradually decreased. This phenomenon was also detected in HeLa cells24. Our study is the first to show that MCM6 and MCM7 are still bound to chromatin in the G2/M phase  and demonstrated that MCM7 is always expressed throughout the whole cell cycle. Directly inferring from ICH results, it is surprising that MCM7 have continued expression throughout the entire M phase Fig. . Immunof25. In this research, we demonstrate that MCM7 depletion will lead to CDK1 inactivation and accelerate mitotic exit  is the key to control cytokinesis. When spindle assemble completely, SAC will be inactive and ultimately leads to CDK1 inactivity. CDK1 can inhibit separase before anaphase. Separase triggers anaphase by cleaving its substrate cohesin, which is the protein that bind sister chromatids. The inactivation of Cdk1 will activate separase and then trigger cohesin degradation, and ultimately initiates the final separation of sister chromatidsxit Fig. . Thus, iBased on this study, we conclude that MCM7 roles are more than just assembling pre-RC complex for launcing DNA replication. MCM7 maintains expression and association with chromatin in M phase to prevent premature exit from mitosis.2.Hepatocellular carcinoma HepG2, human lung carcinoma SPC-A1 and normal hepatocytes LO2 cells were routinely maintained in DMEM medium . Human bladder cancer UMUC-3 was routinely maintained in MEM medium . The medium contains 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were cultivated at 37 °C in a balanced air humidified incubator with an atmosphere of 5% CO26. Briefly, cells were treated with 2 mM thymidine (purchased from Sigma-Aldrich) for 19 h, incubated in fresh medium containing 10% FBS for 9 h and then synchronized for an additional 18 h with 2 mM thymidine. After washing three times with PBS, the blocked cells were incubated in fresh medium containing 10% FBS. Cells were harvested at 0, 3, 6, 9, 12 and 21 h time points. The cell-cycle progression was detected by flow cytometric analysis. For mitosis block, cells were treated with 0.2 μg/ml nocodazole (purchased from Sigma-Aldrich) for 12 h. After washing three times with PBS, the blocked cells were incubated in fresh medium containing 10% FBS. Cells were harvested at 0, 1, 2, 3 and 4 h. The cell-cycle progression was detected by flow cytometric analysis.Cells were synchronized at G1/S border by double thymidine blockTotal proteins from cells were extracted using ice-cold lysis buffer  for 5–10 min, then centrifuged at 12000 rpm form 10 min at 4 °C to obtain the whole cell lysate. Proteins (about 20 μg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes and incubated overnight at 4 °C with antibody against MCM4, MCM5, MCM6, and MCM7 (Santa Cruz), p-histone H3 (Thr3) , histone H3 , p-Cdk1 (Abcam) or GAPDH (Bioworld technology). After washing with Tris-buffered saline with 0.1% Tween 20, the membranes were incubated with HRP-conjugated IgG at room temperature for 1 h. Signal detection was carried out with an ECL system . WB band were quantified with gel-pro software and expressed as optical density fold difference related to GAPDH (relative OD).2, 50 mM Na4P2O7, 0.1 mM NaVO3, 0.5% triton X-100) containing protease inhibitors, then set on ice for 5–10 min for incubation. The tubes were flicked to mix the solution every 2–3 min during incubation. Subsequently, 30% ice-cold sucrose containing protease inhibitors was added to the bottom of the tubes. The tubes were then spinned at 12–15 rcf, 10 min, 4 °C and the supernatants were transferred to new tubes. The pellets were washed with EB buffer and flicked to dislodge the pellets from the wall of the tubes and vibrated briefly for resuspension, followed by spinning in a microfuge, 12–15 rcf, 5 min, 4 °C. Combined the supernatants from the two steps . The pellets were resuspended with EB buffer (the pellets are the chromatin binding fraction) and analyzed by Western blot.Cells were harvested and resuspended in tubes with EB buffer  paraformaldehyde (PFA)/PBS after treatment and then permeabilized for 15 min in 0.25% (v/v) Triton X-100/PBS. After fixation and permeabilization, cells were washed three times in PBS and then blocked with goat serum for 1 h. Cells were incubated with antibodies against MCM7 (Santa Cruz) as required for 4 °C overnight, followed by three times wash with PBS and a 60 min incubation with goat anti-mouse HRP-conjugated secondary antibody. Positive signals were visualized using 3, 3′-diaminobenzidine.Cells grown on 6-well plates were fixed for 15 min in 4% (w/v) para-formaldehyde (PFA)/PBS after treatment and then permeabilized for 15 min in 0.25% (v/v) Triton X-100/PBS. After fixation and permeabilization, cells were washed three times in PBS and then blocked with goat serum for 1 h. Cells were incubated with antibodies against MCM7, β-Tubulin (Santa Cruz) as required for 4 °C overnight, followed by three times wash with PBS and a 60 min incubation with goat anti-mouse FITC secondary antibody. Fluorescence images were taken under an Olympus fluorescent microscope.Cells were fixed in 70% ethanol overnight at 4 °C. Fixed cells were then washed once in ice-cold PBS and stained with propidium iodide (PI) staining solution  for 20–30 min. PI-stained cells were then analyzed for their DNA content by using FACS .5) were seeded into 6-well plates and then were transfected with siRNA in diluted Lipofectamine containing Opti-MEM Medium (Invitrogen) according to manufacturer’s protocol. Non-targeting siRNA was used as the negative control.Three Cdc6-targeting siRNAs (si-MCM7: 5′GAGTTGGTGGACTCAATTT3′) were purchased from Guangzhou Ribobiotech. For the transfection of siRNA, cells  and statistical significance between different groups was determined by the Student’s t-test. Statistical comparisons were made using one-way ANOVA.


Corneal endothelial cells (CECs) are essential for maintaining corneal stromal hydration and ensuring its transparency, which is necessary for normal vision. Dysfunction of CECs leads to stromal decompensation, loss of transparency and corneal blindness. Corneal endothelium has low proliferative potential compared to surface epithelial cells leading to poor regeneration of CEC following injury. Additionally, the tissue exhibits age related decline in endothelial cell density with re-organisation of the cell layer, but no regeneration. The mechanisms which control proliferation and differentiation of neural crest derived CEC progenitors are yet to be clearly elucidated.Prdm gene family which are specifically expressed in human (h) CECs with a view to begin addressing their potential roles in CEC biology, focussing especially on Prdm 4 and 5 genes. By performing semi-quantitative reverse transcription coupled to PCR amplification we found that in addition to Prdm4 and Prdm5, Prdm2 and Prdm10 genes are expressed in hCECs. We further found that cultured primary hCECs or immortalised HCEC-12 cells express all of the Prdm genes found in CECs, but also express additional Prdm transcripts. This difference is most pronounced between Prdm gene expression patterns of CECs isolated from healthy human corneas and immortalised HCEC-12 cells. We further investigated Prdm 4 and Prdm 5 protein expression in cultured primary hCECs and HCEC-12 cells as well as in a human cadaveric whole cornea. Both Prdm 4 and Prdm 5 are expressed in human corneal endothelium, primary hCECs and in HCECs-12 cells, characterised by expression of the Na+/K+-ATPase. We observed that both proteins exhibit cytosolic  as well as nuclear localisation within the endothelial layer, with Prdm 5 being more concentrated in the nuclei of the endothelial cells than Prdm 4. Thus, our work identifies novel Prdm genes specifically expressed in corneal endothelial cells which may be important in the control of CEC differentiation and proliferation.Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifiers are important for driving differentiation of a variety of cellular types. Many Prdm proteins are expressed in specific precursor cell populations and are necessary for their progression to a fully differentiated phenotype. In the present work, we sought to identify members of the  •Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifying genes in corneal endothelial cells (CECs) was undertaken.The analysis of •Prdm 2, 4, 5 and 10 genes were found to be expressed in human CECs.•Prdm 4 and 5 protein expression was characterised in CECs, ex vivo cultured corneal endothelial cells, and the intact cornea. Loss of the endothelial cells leads to the swelling of cornea and loss of corneal transparency . CurrentDrosophila transcription factors Suppressor of variegation 3–9, Enhancer of Zeste and Trithorax (SET) and Positive regulatory domain I-binding factor 1/Retinoblastoma protein-interacting zinc finger gene 1 (PR) motifs originally identified in Prdm1 and Prdm2, respectively) domains, similar to, but distinct from the SET domains of histone lysine methyltransferases (Prdm 5 gene lead to the brittle cornea syndrome (BCS)  . Prdm 5 purposes .Prdm genes in hCECs and whole cadaveric cornea with special focus on the expression of Prdm 4 and 5 genes because of their documented involvement in corneal function. Further, we investigate the expression of Prdm genes in cultured primary hCECs and an immortalised cell line, HCEC-12 and find that both show overlapping, but distinct expression of Prdm genes as compared to CECs or the whole human cornea. Isolated Descemet's membranes together with the attached CECs express Prdm 2, 4, 5, 10 and very weakly Prdm 11 mRNA. Within the whole cornea, we detected Prdm 1, 2, 4, 5 and 10 mRNA expression. Moreover, we demonstrate that two of the selected Prdm proteins, Prdm 4 and Prdm 5, are expressed in both primary hCECs and HCEC-12 cells. Both Prdm 4 and 5 are found in the endothelial cells of the whole cornea. Our results pave the way for further investigations into the roles of these Prdm genes in CEC proliferation, maintenance and differentiation which may be useful for generating CECs for cell transplantation.Given the importance of Prdm proteins in controlling cell fate decisions during development, we reasoned that some of them may regulate various aspects of CEC proliferation and differentiation. To date, no systematic analysis of expression of these genes in corneal endothelial cells has been undertaken. Here, we present the first analysis of expression of all human 22.1® reagent (Invitrogen/ThermoFisher Scientific) was used according to manufacturer's instructions. Human donor corneas were obtained from Bristol and Manchester Eye Banks, UK, with research consent. To isolate total RNA from the DM/Endothelium complex, hCEC and HCEC-12 cells, we used micro-prep RNA extraction kit from Agilent Technologies. A total of 5 × 105 HCEC-12 and primary hCECs were harvested at passage six and two, respectively, following seven days of culturing, for RNA extraction.RNA was extracted from whole human donor corneas, Descemet's membrane (DM)/Endothelium complex, primary cultured human corneal endothelial cells (hCECs) and from cultured HCEC-12 cells. To isolate total RNA from the normal cornea, Trizol® II Reverse Transcriptase was used to perform RT according to the manufacturer's instructions using 1 μg of total RNA and random hexamer primer mix (Invitrogen/ThermoFisher Scientific). Subsequently, 1 μl of cDNA was used for PCR amplification with gene-specific primers. Control reactions were performed without the addition of reverse transcriptase in the reaction mix. Cycling for PCR amplification was performed using the following programme: 94 °C for 2 min followed by 40 cycles of 94 °C for 30 s, 59 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 5 min. PCR products were separated using 2% agarose gel electrophoresis.SuperScriptThe following gene-specific primers were used: Prdm1F 5′ CAGTTCCTAAGAACGCCAACAGG 3’; Prdm1R 5′ GTGCTGGATTCACATAGCGCATC 3’; Prdm2F 5′ TTGGGCTTGCTCAGGAGAAGAG 3’; Prdm2R 5′ GCTGCTATCTCAGGGTTGTCTTC 3’; Prdm3(MDS)F 5′ CCTTATGTGGGAGAGCAGAGGT 3’; Prdm3(MDS)R 5′ GAAGGCTATTCCTACGTCTGAGC 3’; Prdm3(MECOM)F 5′ CCTGCTTCAGATGGTTCCTTGC 3’; Prdm3(MECOM)R 5′ GGTGAAACAAGAATCCTGGAGAAG 3’; Prdm4F 5′ CCAAAGCAGCTTGTTCTCCGTC 3’; Prdm4R 5′ TAGAGGTCCAAAGCAAGTCCGC 3’; Prdm5F 5′ ACTCTGAGGAGAGACCGTTCCA 3’; Prdm5R 5′ AGCATCGCAGTGATGGCACTTG 3’; Prdm6F 5′ GTGAGGAACACGCAGCATCTCT 3’; Prdm6R 5′ TTCTCCGCAGTGCCTTGCACAT 3’; Prdm7F 5′ AGACGAAGAGGCAGCCAACAGT 3’; Prdm7R 5′ GCCACCAGGTTCTGCTCTTCAT 3’; Prdm8F 5′ CTGTGTCCTGAGCCATACTTCC 3’; Prdm8R 5′ CCTTCTGAGGAACCATTTGCTGC 3’; Prdm9F 5′ ACGAAGAGGCAGCCAACAATGG 3’; Prdm9R 5′ GCCACCAGGTTCTGCTCTTCAT 3’; Prdm10F 5′ CCGCAAAGACTTCCTGTGTTCC 3’; Prdm10R 5′ GCTGATGCGGTCGGCTTTCTTG 3’; Prdm11F 5′ CCAGGAAACCATTCACCGCAAC 3’; Prdm11R 5′ CCTCAGGTCTTCTGGGTTATCC 3’; Prdm12F 5′ TGCACGTAACGAACAGGAGCAG 3’; Prdm12R 5′ GTGAGTTTCCGTACCACACCAG 3’; Prdm13F 5′ CTAACTCCTTGGCTCAGTGGTTC 3’; Prdm13R 5′ CAGTACCAGCAGATGTAGCGCT 3’; Prdm14F 5′ CCTTGTGTGGTATGGAGACTGC 3’; Prdm14R 5′ CTTTCACATCTGTAGCCTTCTGC 3’; Prdm15F 5′ GGCACTTGTGAGAAGACCTTCC 3’; Prdm15R 5′ GAGGTTGCTGTTGGTGGAGAAG 3’; Prdm16F 5′ CAGCCAATCTCACCAGACACCT 3’; Prdm16R 5′ GTGGCACTTGAAAGGCTTCTCC 3’.2.2Primary hCEC culture from peripheral DM with endothelial cells  was initiated via primary explant culture. DM was cut into small pieces with fine scissors and placed into a T25 cell culture flask. Cells were initially maintained in 1 ml basal medium OptiMEM-I supplemented with 8% Fetal Bovine Serum (FBS), 1% L-Glutamine, 1% penicillin/streptomycin . Small amount of medium was used in order to allow the pieces of the peripheral DM to come in contact with the bottom surface of the flask and to promote cell outgrowth into the flask. The culture medium was not exchanged until primary cell outgrowth occurred, usually 7–14 days after culture initiation. After that, the culture medium was exchanged every 2nd day until the culture reached confluence. After reaching confluence the primary culture was sub-cultured using 0.05% trypsin/EDTA.2. Confluent cultures were split 1:2 to 1:4 every 4–5 days using 0.05% trypsin/EDTA. All cell cultures were kept in a humidified Heracell ™ incubator at 37 °C, 5% CO2, 95–98%.HCEC-12 cells, purchased from The Leibniz Institute DSMZ , were cultured using F99 (mixture of Ham's F12 and Medium 199 (ThermoFisher Scientific) in 1:1 ratio) medium supplemented with 10% FBS. The medium was changed every 2 days. The cells were seeded out at the density of 50 cells/mm2.32 density for 24 h prior to fixing and processing for immunocytochemistry. The cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilised using blocking solution  in PBS) supplemented with 0.1% v/v Triton X-100 for 20 min at room temperature. Following permiabilisation, cells were incubated in blocking solution for one hour at room temperature. Primary antibodies were added to the fixed cells in blocking solution overnight at 4 °C, followed by the incubation with appropriate secondary antibodies. The following primary antibodies were used: rabbit anti-Prdm4 antibody, Abcam (ab156867), rabbit anti-Prdm5 antibody, Abcam (ab79016) and rabbit anti-Prdm5, Abcam antibody (ab84572), all at 1:500 dilution, and mouse monoclonal anti-alpha 1 Na+/K + - ATPase antibody, Abcam (ab7671) at a 1:100 dilution. Secondary goat anti-rabbit and anti-mouse antibodies conjugated to Alexa 488 or Alexa 568 (Invitrogen/ThermoFisher Scientific) were used at 1:1000 dilution together with Hoechst DNA stain prior to mounting immunolabelled cells in DAKO mounting medium. Immunolabelled cells were visualized using the Leica Microsystems CMS confocal microscope.HCEC-12 and primary hCECs were cultured in glass chamber slides, coated with 0.1 mg/ml poly-D-lysine (PDL) (Sigma Aldrich), at 5000 cells/cm2.4Prdm4 antibodies or Epitope retrieval solution 2 (pH9.0) (AR9640) for anti-Prdm5 antibodies. Both antibodies were used at 1:500 dilution. The staining programme used was as follows: 1) peroxide block - 5min, 2) primary antibody was applied for 30 min, 3) polymer HRP was added for 8 min, 4) mixed DAB was added for 10 min. Sections were counterstained with haematoxylin for 5 min to visualise nuclei.Cornea were fixed in formalin and embedded in paraffin for processing using LEICA Bond-III automated system with LEICA Bond Polymer Refine Detection kit (DS9800) according to the manufacturer's instruction. Bond Polymer Refine Detection kit is a biotin-free, polymeric horseradish peroxidase (HRP)-linker antibody conjugate system for the detection of tissue-bound mouse and rabbit IgG's. All sections were subjected to antigen heat retrieval at 100 °C for 20 min prior to staining procedure using either Epitope retrieval solution 1 (pH 6.0) (AR9961) for anti-33.1Prdm genes. The corneas obtained were from deceased 64 year old female, 69 year old male and 87 year old male.To begin our investigations we initially utilized cadaveric whole human cornea to assess expression profiles of all human Prdm genes was assessed using gene-specific primers. We detected expression of five Prdm mRNAs out of a total of 17 primer combinations tested. Prdm 1, 4 and 5 were expressed at relatively high levels, while Prdm 2 and 10 showed moderate to low expression levels in total normal human cornea  express Prdm 2, 4, 5, 10 and very weakly 11 , Prdm 6, 8, 11 and 16 , 6, 8 and 16 were detected in HCEC-12 cells by RT-PCR in addition to those observed in DM/CECs tissue preparations. Given that HCEC-12 cells are immortalised using the early region of SV40 genome containing the large T antigen and the small t-antigen, they may be re-programmed to express other Prdm genes as part of their phenotype. For example, Prdm 3 (Mecom form) has a well-documented role in myeloid leukemogenesis (Prdm 16 in humans lead to haematological malignancies (Prdm 1, 2, 3 and 16 genes encode two isoforms, those which contain the PR/SET domain and those which lack it, and the biological roles of these two isoforms in controlling cellular proliferation are opposite, with the PR/SET-containing isoforms being anti-proliferative and the PR/SET-lacking isoforms supporting cellular proliferation in a variety of cells (Prdm genes in various cell lines as well as in normal cornea and primary hCECs in the future in order to understand their potential roles in controlling CEC proliferation and differentiation.Immortalised HCEC-12 cells showed marked differences in the expression profile of detectable ogenesis  where itogenesis . Conceivogenesis  and its ogenesis . Furtherogenesis  and somegnancies . Therefoof cells . TherefoPrdm genes in human cornea with the special emphasis on the corneal endothelial cells with the view of identifying those Prdm genes which may be important for CEC biology. Of the Prdm genes identified in our screen, we further analysed expression of Prdm 4 and 5 proteins in intact corneas as well as primary hCECs and HCEC-12 cell line. We found that Prdm 4 and 5 are expressed in both corneal endothelial and epithelial cells with slightly different subcellular distribution of these two proteins within the above cells. The remaining Prdm genes identified in this screen will warrant further study to understand their contribution to the biology of CECs. We focussed on Prdm 4 and 5 genes in part because there is evidence which implicates both of these proteins in corneal biology. The role of Prdm 5 is by far better understood, while Prdm 4 is clearly a novel candidate with potentially important roles in corneal biology. Indeed, it is especially intriguing, given that Prdm 4 was initially cloned in a screen for p75NTR interacting proteins (In this report we focussed our attention on the characterisation of expression of proteins , as p75Nproteins . It will5Prdm 2, 4, 5 and 10 genes are expressed in hCECs and in the whole human cornea. Moreover, Prdm 4 and 5 proteins are found in the CECs of the cornea where they are distributed both in the nucleus and the cytosol. Identification of these in part novel genes, which are important in controlling cellular proliferation and differentiation, adds to the repertoire of potentially important players in helping the generation of CECs for therapeutic purposes in future studies.We present evidence that BBSRC grant BB/J006602/1. KR and MR were supported by a PhD studentship from Anglia Ruskin University and the Vision and Eye Research Unit REF2014 QR fund. The funders did not participate in the work or data processing.AC and CG were supported by the


BmαTX14 is an α-neurotoxin extracted from the venom gland of the East Asian scorpion Buthus martensii Karsch and can affect ion channel conductance. Here, we investigated the functions of rBmαTX14 using the Caenorhabditis elegans model. Using western blot analysis, rBmαTX14 was shown to be expressed both in the cytoplasm and inclusion bodies in the E.coli Rosetta (DE3) strain. Circular dichroism spectroscopy analysis demonstrated that purified rBmαTX14 retained its biological structures. Next, feeding nematodes with E.coli Rosetta (DE3) expressing rBmαTX14 caused extension of the life span and promoted the locomotion of the nematodes. In addition, we identified several genes that play various roles in the life span and locomotion of C. elegans through microarray analysis and quantitative real-time PCR. Furthermore, if the amino acid site H15 of rBmαTX14 was mutated, rBmαTX14 no longer promoted the C. elegans life span. In conclusion, the results not only demonstrated the functions and mechanism of rBmαTX14 in C. elegans, but also provided the new sight in the utility of recombinant peptides from scorpion venom.The scorpion has been extensively used in various pharmacological profiles or as food supplies. The exploration of scorpion venom has been reported due to the presence of recombinant peptides. r Scorpions, which are considered ‘living fossils’, maintain primary Paleozoic scorpions features such as the venom apparatus, book lung and pectin . AlthougBmαTX14, a peptide extracted from East Asian Buthus martensii Karsch, is known to be a potent blocker of the Na+ currents of root ganglia neuron. The rBmαTX14 cDNA sequence was obtained from the BmK cDNA library [Pichia pastoris and in E.coli [Scorpion venom contains many polypeptides, usually several amino acid residues compiled into long single chains –10. Thes library , and then E.coli , 17.C. elegans to assess rBmαTX14 function. C. elegans was the first multicellular organism to have its entire genome sequenced, and approximately 35% of C. elegans genes have human homologs). Additionally, this small soil nematode has a short life cycle [C. elegans a unique model, especially for life span study and disease analysis [C. elegans model system to investigate the bioactivity of rBmαTX14, and the results show that this polypeptide plays potential roles against aging and promotes locomotion.In this work, we used the animal model fe cycle , 19. Allanalysis , 21. In BmαTX14 DNA sequence was amplified via the Polymerase Chain Reaction (PCR) with the forward primer CCCATATGGTTCGGGATGCT and the reverse primer CGGGATCCTCAATGGCATTT. After digestion with the Nde I and BamH I restriction enzymes , the rBmαTX14 DNA sequence was ligated into the pET28a vector with T4 DNA ligase . The insertion accuracy was verified by DNA sequencing . The antibody was obtained from Proteintech .The rBmαTX14 was “VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGKYGNACWCIKLPDDVPIRVPGKCH”, and then the cDNA sequence of the negative control for BmαTX14 was created according to the sequence of BmaTX14. The protein sequence of this negative control was “VCHGKPRVPIDVPDKLIWCCNAYGGKWGCQGYEASNGTDLCNKGCNEATHYCNVEPCAKYIDRA”. So the random protein was expressed as the negative control using pET28a-rBmαTX14 (NEG).Since the protein sequence of C. elegans strain N2 (wide type) and E. coli strain OP50 were obtained from the Caenorhabditis Genetics Center at the University of Minnesota. C. elegans were grown at 20°C on nematode growth media (NGM) plates and were propagated on E. coli OP50 using standard methods [The  methods . SynchroBmαTX14 plasmid (with 6-His-Tag) or the empty pET28a vector was transformed into competent E.coli strain Rosetta (DE3) cells . Then, E.coli cells were maintained at 37°C in Luria–Bertani medium with vigorous shaking. Isopropyl-β-D-thiogalactopyranoside  was added at a concentration of 1 mM when the OD600 of the E.coli reached 0.4. After further incubation at 28°C for 4 h, the cells were harvested for further use.The recombinant pET28a-rE.coli cells were harvested and lysed with ultrasonication, and the lysate was centrifuged at 4,000 rpm for 10 minutes at 4°C. Then, the supernatant was centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was obtained from the cytoplasm, while the insoluble fraction primarily included inclusion bodies. The supernatant and insoluble fractions were lysed again for 10 minutes at 100°C. Then, the protein samples were analyzed by western blotting.The BmαTX14 inclusion bodies were lysed in denaturation solution . After 2 h of incubation, the solution was slowly added to 100-fold volume of renaturation solution  and incubated at 16°C for 24 h. The precipitate was removed by centrifugation at 12,000 rpm for 15 min. The supernatant was desalted and concentrated with a centrifugal filter device  at 5,000 g for 4 h. Next, 0.1% TFA was added to the concentrated protein solution to remove the precipitate, and the supernatant was injected into RP-HPLC. Renatured rBmαTX14 was purified by RP-HPLC on a C18 column  (Elite-HPLC) using a linear gradient of 5–95% acetonitrile with 0.1% TFA in 60 min at a constant flow rate of 5 ml/min, and the protein was detected at 230 nm. The rBmαTX14 peak appeared at 21 min and was manually collected.The r2O was measured in the 195–250 nm wavelength range (protein secondary structure) at 25°C on a Jasco J-810 spectropolarimeter  with a 0.1 cm pathlength cylindrical cell. The bandwidth was 1 nm, and the response time was 1 s. All the samples were allowed to equilibrate thermally for 5 min prior to the CD measurements. Each sample spectrum was corrected by subtraction from the spectrum (baseline) that was recorded for H2O. Each spectrum is an average of three different scans that were obtained by collecting data at 0.1 nm intervals at a scan speed of 200 nm/min.The 20 μM peptide far-UV CD spectra in HE. coli OP50 , 50 μg/mL carbenicillin and 0.1 μg/mL fungizone with gentle shaking until L4. Next, 2′-deoxy- 5-fluorouridine  was added to prevent progeny growth. After further incubation for 24 h, culture aliquots were dispensed into plate wells , and appropriate volumes of E.coli solution were added to achieve the indicated concentrations. The total incubation volume was 100 μL per well, and the plates were sealed with Parafilm to prevent evaporation. The day of nematode transfer was counted as day 0 in the life span analysis. The fraction of nematodes alive was scored microscopically every 2 days. Survival data were analyzed using the log-rank test using SPSS 17.0 for Windows.All life span assays were conducted in 96-well plates using liquid culture at 20°C as previously described . BatchesE. coli strain rBmαTX14 or the control with initial OD600 of 0.6–0.7) with gentle shaking (110 rpm) at 20°C. Regarding the food clearance assay, the plates were measured every day using the microplate spectrophotometer for a week as previously described [Newly synchronized L1 nematodes were incubated in 96-well plates containing foods . The results were analyzed using the Molecule Annotation System (MAS) 3.0 .Total RNA was extracted from adult worms grown at 20°C on NGM plates using TRIzol™ Reagent  according to the manufacturer’s instructions. We further examined the gene expression changes through DNA microarray expression profiling. Affymetrix Genechips were used to perform Waltham, USA) according to the manufacturer’s instructions. Act-1 expression was used as the control reference. Quantitative RT-PCR was conducted using SYBR Green PCR Master Mix  and analyzed with the 7500HT Fast Real-Time PCR machine .The mRNA was converted to cDNA using the RevertAid First Strand cDNA Synthesis Kit  was consumed in 7 days was approximately the same in the rBmαTX14 and control group  analysis. The results showed that 12 out of the 16 tested genes showed similar changes using qRT-PCR and microarray analysis . The priwww.capitalbio.com). With the MAS 2.0 tool, the pathways are ranked by statistical significance by calculating their p-values based on the hypergeometric distribution [Moreover, all 190 differentially expressed genes were analyzed using a free Molecular Annotation System 2.0  increased the median day of life span by 14.8% compared with empty vector and pET28a-rBmαTX14 (H15⟶F15). P-value indicated this change was statistically significant. Specifically, the life span with pET28a-rBmαTX14 and pET28a-rBmαTX14 (T18⟶R18) were approximately 50 days in length, while the life spans with empty vector and pET28a-rBmαTX14 (H15⟶F15) were approximately 44 days in length  (BmαTX14 and pET28a-rBmαTX14 (NEG) all have inclusion bodies, so calorically/nutritionally restricted or biomass should not be the main reason that affects gene transcription. Maybe the peptides were digested by proteases in the gut and absorbed by the gut of the animal. Though BmαTX14 is known to be a potent blocker of the sodium channel, genome sequence analysis showed that voltage-gated sodium channel was absent in C. elegans. In the muscle cells of C. elegans, voltage-gated calcium channels had the similar functions as well as the sodium channel [BmαTX14 is through the related voltage-gated channel?Since starvation could elongate the life span of nematodes , caloric14 (NEG) . Because channel –54. ThenBmαTX14. Feeding the nematodes with pET28a-rBmαTX14, we’ve demonstrated that rBmαTX14 caused extension of the life span and promoted the locomotion of the nematodes. Further investigation uncovered the specific genes that play various roles in the life span and locomotion of C. elegans. In addition, the amino acid site H15 of rBmαTX14 was proved to be an important site in the function of the protein. This interesting finding may provide an insight into the utility of scorpion venom in anti-aging as food or medicine.Overall, in this study, we’ve expressed, purified and measured the secondary structure of recombinant protein rS1 FigBmαTX14 (NEG) did not increase the C. elegans life span. The nematodes were treated with empty vector, pET28a-rBmαTX14 and pET28a-rBmαTX14 (NEG) in 96-well plates. The survival data were plotted using the Kaplan-Meier method and analyzed by log-rank tests using SPSS 17.0 software. Body bend frequency (B) and reversals (C) were analyzed using a SAMSUNG SCC-101BP device and VideoMach software . 100 worms were observed for each condition in motility assays, and the data represent an average of at least three independent experiments. (D) Expression of rBmαTX14 and the negative control in E. coli. Lanes 1 and 3 indicated cell lysate from E.coli with pET28a, lanes 2 and 4 indicated cell lysate from E.coli with pET28a-rBmαTX14, and lane 5 and 6 indicated cell lysate from E.coli with pET28a-rBmαTX14 (NEG). The primary antibody utilized was anti-6-His.(A) pET28a-r(TIF)Click here for additional data file.S1 TableC. elegans fed with empty vector, and Group 2 is C. elegans fed with pET28a-rBmαTX14.Group 1 is (DOC)Click here for additional data file.S2 TableGene lists were for 171 Up and 11 Down regulation genes.(DOC)Click here for additional data file.S3 TableC. elegans fed with control E.coli strain with empty vector, Group 2 is C. elegans fed with pET28a-rBmαTX14, Group 3 is C. elegans fed with pET28a-rBmαTX14 (H15⟶F15), and Group 4 is C. elegans fed with pET28a-rBmαTX14 (T18⟶R18).Group 1 is (DOC)Click here for additional data file.S4 TableBmαTX14 and pET28a-rBmαTX14 (NEG) were starting from L4.Life span analysis of empty vector, pET28a-r(DOC)Click here for additional data file.


Polyfuran films produced by electropolymerization of a series of oligofurans substituted with alkyl groups show improved properties, such as good conductivity and stability, well-defined spectroelectrochemistry and smooth morphology. –1, good environmental and electrochemical stabilities, very smooth morphologies (roughness 1–5 nm), long effective conjugation lengths, well-defined spectroelectrochemistry and electro-optical switching (in the Vis-NIR region), and have optical band-gaps in the range of 2.2–2.3 eV. A low oxidation potential needed for polymerization of oligofurans (compared to furan) is a key factor in achievement of improved properties of polyfurans reported in this work. DFT calculations and experiments show that polyfurans are much more rigid than polythiophenes, and alkyl substitution does not disturb backbone planarity and conjugation. The obtained properties of polyfuran films are similar or superior to the properties of electrochemically prepared poly(oligothiophene)s under similar conditions.Polyfurans have never been established as useful conjugated polymers, as previously they were considered to be inherently unstable and poorly conductive. Here, we show the preparation of stable and conducting polyfuran films by electropolymerization of a series of oligofurans of different chain lengths substituted with alkyl groups. The polyfuran films show good conductivity in the order of 1 S cm Polyfuran (PFu), on the other hand, has scarcely been explored and has not been established as a conductive polymer. This is because of the difficulty in polymerization, the low stability and the poor properties of the polyfuran samples that have been obtained.5 The reported electrical conductivities of doped polyfurans were measured in the range of 10–5 to 10–2 S cm–1, which is significantly lower than the conductivity of polythiophenes electrochemically prepared from a terthiophene (PT3) (100 S cm–1) and thiophene monomers (102 S cm–1). Polyfurans have also been reported as environmentally and electrochemically unstable. For example, the authors noticed that upon exposure to ambient light and air, films of chemically prepared alkylated polyfuran bleached over a period of hours to days.9 Furthermore, the electrochemical redox activity of polyfuran was shown to degrade during cycling in acetonitrile (ACN) solution.10The field of conjugated and electrically conducting polymers continues to attract much attention both scientifically and technologically. The main problem associated with electrochemical polymerization is the high oxidation potential required to polymerize furan, which results in irreversible over-oxidation of the resulting polyfurans. A mixture of trifluoride diethyl etherate and ethyl ether (BFEE/EE) as a solvent was used to reduce this electropolymerization potential. However, the reported conductivities of the resulting polyfurans were small (up to 10–2 S cm–1), and their absorption spectra (λmax = 420 nm)11 reveal a short conjugation length of about eight furan rings.14 Kanatzidis et al., used terfuran as the starting monomer to lower the potential needed for polymerization to less than 1.0 V (vs. SCE).11 The resulting polyfuran was characterized by a variety of techniques and showed a red shifted absorbance maximum centered at 468 nm. Nevertheless, the polyfuran produced had a low conductivity of 2 × 10–3 S cm–1, which is probably due to defects in the polymer, as were observed by IR spectroscopy. Chemically synthesized polyfuran did not appear to be highly conjugated because of a significant degree of furan ring-opening defects. It is noteworthy that in contrast to polyfurans, the incorporation of furan blocks in conjugated polymers resulted in high quality materials.16 Overall, despite numerous reports on the preparation of polyfuran ,5 it appears that there are no reports of polyfurans with properties such as high conductivity, stability, high conjugation length and small number of defects, all of which are required for their application as organic electronic materials.Many researchers have tried to overcome preparation problems by varying the conditions of polymerization.5-Fu9) and recently alkyl substituted α-oligofurans  and Fu16-6C6) that exhibit higher fluorescence, better packing, greater rigidity, and higher solubility than the corresponding oligothiophenes. We also showed that these oligofurans display good electronic properties, e.g., field effect mobilities and on-off ratios, similar to those of the corresponding oligothiophene analogues.18 Thus, we surmised that polyfurans would not only possess similar properties, but would have advantages over other conductive polymers. The rigidity of their backbone is expected to enable the introduction of large side substituents without breaking planarity and π-conjugation. Moreover, polyfurans can be synthesized from renewable resources19 and may be biodegradable.20 Thus, we believe that the reported poor properties, such as low conductivity,5 blue-shifted absorption,11 and instability,9 of polyfurans were due to defects produced during their preparation rather than intrinsic properties of polyfurans.We previously reported the preparation of long unsubstituted α-oligofurans  with tributyl(furan-2-yl)stannane (9), -5-yltributylstannane (10), and -5-yltributylstannane (11), respectively. The precursor 2,5-dibromo-3-octylfuran (12) was prepared by bromination of 3-octylfuran with N-bromosuccinimide (NBS) with a 68% yield.Oligomers 1–5  and 2 we1–5 Cl2 gave 3,3′-dioctyl-2,2′-bifuran (15) with a 74% yield. Stille coupling of dibromo derivative 16 (obtained from the bromination of 15 using NBS) with -5-yltributylstannane (11) yielded dioctyl-substituted octafuran 5.For the synthesis of octafuran 5 , dibromo2 has the highest octyl chain to furan ratio (1 : 3), while oligomer 4 has the lowest octyl chain to furan ratio (1 : 7). In order to synthesize polymers having at least a 1 : 1 ratio of alkyl chain to furan ring, bromo groups present in compound 14 were replaced with alkyl chains. Kumada coupling of 14 with methylmagnesium bromide gave compound 6 (7 (18 followed by coupling with CuCl2.Oligomer mpound 6  with a mund 6 (7  was syntca. 1.8 V vs. SCE), at which point ring opening and irreversible degradation leads to non-conjugated and low quality polyfurans. Thus, the electropolymerization potential needs to be lowered. This is achieved by using oligofurans rather than furan monomer as the repeat units. Starting from terfuran, the oxidation potential of oligofurans drastically decreases, and the polymerization process proceeds below 1.0 V vs. SCE.11 Such low oxidation potentials allowed the preparation of polyfurans reported herein under air at ambient conditions, which can be beneficial for their possible applications.Electropolymerization of furan monomer could be achieved only at high potentials . It is worth noting that the electrodes were not modified and specially cleaned as they were in the preparation of smooth polythiophenes.24 Representative cyclic voltammograms (CV) for the polymerization of P3 (PFu5-C8) on ITO electrode, showing stepwise increases in the current over the course of polymerization, are displayed in The electrodeposition of polymers 1, 2, 4 and 5 , measured from the first CV curve of the polymerization, were used for comparison between different oligomers (Eox for P1 (PFu3) is 0.62 V, whereas for P2 (PFu3-C8) and P3 (PFu5-C8), it is 0.51 V and 0.43 V, respectively. Furthermore, polyfurans are obtained at lower oxidation potentials than polythiophenes, as shown by the oxidation onset potential of terfuran, which is 0.33 V lower than that of terthiophene.Oxidation peak potentials are poorly resolved for oligomers 5 Fig. S3. Therefoligomers . As expe We found that TBACF3SO3/ACN electrolyte .CVs of polyfurans in monomer-free TBACFon peaks  and S5‡,P1–P7 films on ITO electrodes were performed to evaluate their electronic structure. All prepared polymers exhibited well-defined spectroelectrochemical responses, namely, a reversible and distinct change in the absorption spectrum during the redox processes (doping/dedoping). P1, P2, and P6 films  states.26 Only the 0.7 V spectral trace of P6 , which is partially offset by an increase in the lowest occupied crystal orbital (LUCO) energy (i.e. the energy of the conduction band). Consequently, the oxidation potential of alkyl-substituted polyfurans decreased. The increase of the HOCO energy is consistent with the electron donating property of alkyl groups.The optical band gaps of polyfurans estimated from the absorption spectra  for unsuand gaps  and S3‡.and gaps  showed t27 The presence of vibronic coupling may indicate that the polymer backbone is highly rigid, planar and effectively π-conjugated. We have shown that oligofurans exhibit high rigidity, as evident from calculated twisting potentials which are higher than for oligothiophenes. Similar computational results have been recently shown for polyfurans.29 Consequently, the π-conjugation and planarity of polyfurans should not be distorted by various substitution of the polyfuran backbone as strongly as it is in the polythiophene family. Indeed, DFT geometry optimization (PBC/B3LYP/6-31G(d)) of polyfurans P1–P7 shows that the polyfuran backbone remains planar upon alkyl substitution , carbonyl C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O bond (∼1720 cm–1), and O–H bond (∼3500 cm–1). The measured ATR-FTIR spectra of P1 and P3 films on Pt-foil  microelectrode34 or two-band electrode35 have found wide use.36 However, obtaining the absolute value of polymer film conductivity is not straightforward because it depends on many factors that are hard to define, such as non-trivial geometrical factor of the electrode, non-linear dependence of the conductance on film thickness, and undefined contact resistances. Thus, we report conductance values in comparison to that of poly(terthiophene) (PT3), which was electrochemically prepared and measured under the same setup. Additionally, we compare the conductance values of our polyfurans to those of drop-cast commercial poly poly(styrenesulfonate) (PEDOT:PSS) film on the same type of electrode. The conductivity of electrochemically prepared PT3 film7 and that obtained from commercial PEDOT:PSS37 is estimated to be on the order of 1 S cm–1.The conductivity of conjugated polymers strongly depends on many factors, particularly the method of preparation, the presence of impurities, humidity, and the measurement technique.P1–P4, P6 and P7 films. All obtained polyfurans show maximum conductances comparable to those of PT3 and PEDOT:PSS films  for comparison between polymers. As evident from Ehc depends on the degree of alkyl substitution, i.e., Ehc decreases with increasing degree of alkyl substitution  as bipolarons are formed. However, this behavior is not observed in polyfurans P1–P4 due to degradation of the polyfuran films at potentials above 0.8 V, where their conductivity decreases irreversibly.We observe that the conductivity of fully methylated polyfurans 40 Electrochemical degradation of conductive polymers, such as polypyrrole, polythiophene, and polyaniline, has been studied extensively.40 However, very little has been reported on the electrochemical degradation of polyfuran.The electrochemical stability of conducting polymers is of utmost importance for their use in electrochemical systems, such as batteries, electrocatalytic materials, sensor devices, and other electronic components.vs. Ag/AgCl-wire in a monomer-free TBACF3SO3/ACN electrolyte solution for up to 2000 cycles in the air under ambient conditions . In order to properly compare the stability of polyfurans with that of PT3, the potential scan window was shifted by 0.2 V towards higher potentials. The reasoning for this stems from the fact that PT3 has a higher oxidation potential. Under these conditions, polyfurans showed slightly better stability than PT3 , within which the degraded and non-electroactive polyfurans show no absorbance. The stability of optical switching was evaluated by applying repetitive potential pulses (10 s length) of 0.0 V and 0.7 V for polyfurans, and 0.0 V and 0.9 V for PT3 for prolonged time (2 h)  . For comi.e., difference in transmittance of undoped and doped state) obtained after 120 min of cycling  were found to be 39%, 45%, and 51% for P1, P3 and PT3 of the starting activity, respectively. Thus, polyfurans P1 and P3 showed only slightly lower stabilities compared with PT3, but are much better than that reported for a PFu/PEDOT device.10The degraded optical activities  in air under ambient conditions  needed for oxidation (doping), which in turn, is expected to break the conjugation of the backbone via side reactions.42 Thus, the lower potential required for doping the polyfurans is advantageous because it minimizes the occurrence of backbone degradation under ambient conditions.As shown in 1 Controlled film thickness, surface smoothness, and uniformity are important considerations for application of conductive polymers in thin film devices , including photovoltaic cells and OFETs. Electrochemical deposition has some advantages over solution processing of conjugated polymers. It combines polymer formation and deposition in one-step and bypasses the need for polymer isolation and purification. As such, it has found use in fabrication of organic electronic devices.43 However, it is difficult to obtain smooth and homogeneous thin films by electropolymerization, necessitating the optimization of electrodeposition.1 For example, the root mean square roughness (rrms) of PT3 on ITO is 173 nm  was proposed to obtain thin films of less than 100 nm with controllable thickness.46 The drawback of this method, however, is that prior polymer synthesis is necessary.Polymer morphology has a significant effect on polymer charge–transport properties. Fig. S18 and PT oP3 on HOPG electrode .47 Additionally, polyfuran films have high adhesion to the electrode surface and are not easily detached. For example, they cannot be pulled off by sticking adhesive tape to the electrode, as in case of poly(3-methylthiophene) films.48 Additionally, films were found to be completely insoluble in organic solvents, such as THF and toluene.As can be seen in P3 linearly depends on the number of CV cycles used in the polymerization , polyfurans of good quality are obtained. By lowering the polymerization potential using long oligofurans as the starting monomers, the polyfurans could be prepared in open air under ambient conditions.Overall, we have shown that by careful selection of starting oligomers and electropolymerization conditions s.7 Thus, we are convinced that the previously reported low conductivities5 are due to preparation problems, and are not an intrinsic property of polyfurans.The measured high conductivity on the order of 1 S cmP1, we experimentally observe a reduction of around 0.08 eV  and Ehc = 0.30 V for PFu3-3C1 (P6)). Furthermore, Ehc for polyfurans is lower than that of PT3 (Ehc = 0.87 V). Therefore, when compared to polythiophenes, a lower oxidation potential of polyfurans serves as an advantage. The lower doping potential minimizes the occurrence of the destructive side reactions . Moreover, oligofurans show lower oxidation potential compared to oligothiophenes, as evidenced by the onset potentials for oligomer oxidation (0.62 V for terfuran and 0.95 V for terthiophene).We have studied the effect of alkyl substitution on the properties of polyfurans. DFT calculations predict reductions of the band gaps, as well as oxidation potentials of polyfurans, with increasing alkyl substitution ratio 50 is no longer mandatory.As shown by DFT calculations spectra  and S11‡3 under similar conditions. This stability is attributed to lower potential needed for doping of polyfurans. The optical switching stability of polyfurans is found to be comparable with that of PT3, and it is much better than the previously reported stability of the optical switching of a PFu/PEDOT device at 415 nm. Previous studies showing optical switching degradation after only 90 s then is likely attributable to the polyfuran's poor quality, as suggested by the low absorption maximum (λmax = 420).10 Therefore, we believe that the earlier reported instabilities of polyfurans are also due to problems in their preparation.We have carefully studied the stability of electrochemical, optical, and conductive properties of polyfurans in open air under ambient conditions. The stability of the redox activity and conductivity over time showed that polyfurans are actually more stable than polythiophene PTca. 50 nm thickness), we observe a smooth thin film covering the whole electrode surface is formed. As the film continues to grow, globular bumps and aggregates are observed on top of the smooth film surface. As a result, the roughness of the surface increases. For instance, in P3 on HOPG, the rrms value was ca. 5 nm for the underlying film and ca. 17 nm for the full scanned area (Fig. S20We have identified two phases of the polyfuran film growth. Initially (up to  Fig. S20. The fori.e., in organic media electrolyte, in ambient environment, without electrode surface modifications and extensive electrode cleaning. This is certainly an advantage for technological applications of polyfurans.It is worth noting that prepared polyfuran films have lower roughness than most other electrochemically-prepared films under similar conditions,Using long oligofurans as precursors, we were able to obtain and thoroughly characterize polyfuran films with different alkyl substitutions that exhibit high stability, are spectroelectrochemically active, show good conductivity in the open air under ambient conditions, and have smooth morphology. These properties are considerably improved from any other known polyfuran prepared electrochemically. Additionally, we showed that polyfurans exhibit high rigidity, which allows many different substituents to be introduced without distorting the planarity and conjugation of the polyfuran backbone. We showed that regioirregular alkyl-substituted polyfurans do not lose their conjugation, and moreover exhibit reduced band gaps.Polymerization of oligofurans using considerable lower potential than parent furan allowed us to obtain stable polyfurans with very good properties. This is in contrast to the previous belief that polyfurans are intrinsically unstable and possess low conductivity. We believe that this work will establish polyfurans as a fundamental series of conductive polymers with important properties, opening up future applications for polyfurans as conductive polymers.


Strain PV-4 was incubated at varying pH with lactate as the electron donor and NO3–/NO2– and N2O as the electron acceptors. When incubated with NO3– and N2O at pH 6.0, transient accumulation of N2O was observed and no significant NH4+ production was observed. At pH 7.0 and 8.0, strain PV-4 served as a N2O sink, as N2O concentration decreased consistently without accumulation. Respiratory ammonification was upregulated in the experiments performed at these higher pH values. When NO2– was used in place of NO3–, neither growth nor NO2– reduction was observed at pH 6.0. NH4+ was the exclusive product from NO2– reduction at both pH 7.0 and 8.0 and neither production nor consumption of N2O was observed, suggesting that NO2– regulation superseded pH effects on the nitrogen-oxide dissimilation reactions. When NO3– was the electron acceptor, nirK transcription was significantly upregulated upon cultivation at pH 6.0, while nrfA transcription was significantly upregulated at pH 8.0. The highest level of nosZ transcription was observed at pH 6.0 and the lowest at pH 8.0. With NO2– as the electron acceptor, transcription profiles of nirK, nrfA, and nosZ were statistically indistinguishable between pH 7.0 and 8.0. The transcriptions of nirK and nosZ were severely downregulated regardless of pH. These observations suggested that the kinetic imbalance between N2O production and consumption, but neither decrease in expression nor activity of NosZ, was the major cause of N2O accumulation at pH 6.0. The findings also suggest that simultaneous enhancement of nitrogen retention and N2O emission reduction may be feasible through pH modulation, but only in environments where C:N or NO2–:NO3– ratio does not exhibit overarching control over the NO3–/NO2– reduction pathways.pH has been recognized as one of the key environmental parameters with significant impacts on the nitrogen cycle in the environment. In this study, the effects of pH on NO Limitations do exist in extrapolating experimental results from axenic culture experiments to ecological contexts; nevertheless, these experiments demonstrated that stimulation of respiratory ammonification at high pH conditions shifted the N2O-generating NO3–-reducing organism to a sink of N2O. Unlike previous observations, lowering of pH to 6.0 did not lead to inhibition of N2O reduction activity in S. loihica strain PV-4, suggesting that the cause of N2O accumulation was due to the kinetic imbalance of the nitrogen oxide reduction reactions, rather than transcriptional or post-transcriptional regulation. Our observations also suggested that the pH effects on nitrogen-oxide dissimilation reactions were not as influential as the effects of C:N or NO2–:NO3– ratios.In this study, S. loihica strain PV-4 was prepared as described previously  and aluminum crimp seals and autoclaved. Wolin vitamin solution was added from a filter-sterilized 200X stock solution after autoclaving (3 (or KNO2) and NH4Cl from sterilized anoxic stock solutions to final concentrations of 560.3 mg/L (5 mM), 101.1 mg/L (1 mM), and 10.7 mg/L (0.2 mM), respectively. The inoculated serum bottles were incubated in dark at 25°C without shaking. After each experiment, final pH was measured to confirm that pH was maintained constant .The medium for cultivation of eviously . Medium solution  per liteoclaving . The med3–, NO2–, NH4+ and N2O were monitored using established analytical procedures. N2O concentration was measured using HP 6890 Series gas chromatograph equipped with a HP-PLOT Q column  and an electron capture detector . Headspace N2O concentration was measured by manually injecting 100 μL headspace sample into the gas chromatograph. The detection limit was approximately 2 ppmv. The total amounts of N2O in the vessels were calculated from the headspace concentrations using Equation 1.Concentrations of NOh is the headspace concentration in moles/L, Va and Vh are the volumes of the aqueous phase and the headspace, respectively, in liters and H is the dimensionless Henry’s constant of N2O. The dimensionless Henry’s constant at 25°C was calculated as previously described -L and HS-NH3 (N)-L kits , respectively, according to the protocols provided by the manufacturer. Aqueous NO3– concentration was determined with Metrohm 863 Basic IC plus ion chromatography system  equipped with a Metrosep A Supp 4-250/4.0 anion exchange column. In presentations of the experimental data, the quantities of the nitrogen species were expressed in μmoles/bottle for convenience in mass balance calculations.Aqueous NOS. loihica strain PV-4 cells were prepared with 5.0 mM lactate and 1.0 mM NO3– as the electron donor and the electron acceptor, respectively. These precultures were incubated until NO3– and NO2– were depleted, and 0.5 mL of the precultures were inoculated to serum bottles with fresh media adjusted to the desired pH  and amended with 5.0 mM lactate and 1 mM NO3– or NO2–. Immediately after inoculation, 0.5 mL of headspace N2 was aseptically replaced with >99.999% N2O . With time intervals determined from preliminary experiments, headspace and aqueous phase samples were extracted for monitoring of NO3–, NO2–, NH4+ and N2O concentrations and cell densities. For determination of N2O concentration, 100 μL of headspace gas was extracted using a 1700-series gastight syringe  and manually injected into the gas chromatograph. Immediately after headspace sampling, 1.5 mL of the aqueous phase was extracted using a disposable 3-mL syringe . In order to avoid pressure loss in the vessels, 1.6 mL of N2 gas was injected before extraction of the liquid samples. After OD600nm of the extracted cell suspension was measured using Genesys 30 visible spectrophotometer , the suspension was centrifuged and the concentrations of NO3–, NO2–, NH4+ and N2O in the supernatant were measured. Incubation was carried out until no further change in the concentrations of the nitrogen species was observed.Precultures of S. loihica strain PV-4 prepared and carried out identically as described above. For each batch culture amended with NO3– and N2O, N2O concentration was monitored to determine three sampling time points  and during the mid-exponential phase . One-half milliliter samples were collected from three biological replicates upon each sampling event. One milliliter of RNA Protect Bacteria Reagent  was immediately added to each of the aliquots and the mixture was immediately centrifuged for 10 min at 5,000 × g. The cell pellets were stored at -80°C until further processing.The samples for transcription analyses of the genes involved in denitrification and respiratory ammonification were extracted at multiple time points during batch cultivation of TM III Reverse Transcriptase . The remaining eluent was used later in a quantitative polymerase chain reaction (qPCR) to confirm the absence of contaminating DNA. After reverse transcription, each sample was treated with RNase H (Invitrogen) to remove traces of RNA. The cDNA solution was diluted five-fold with nuclease-free water and stored at -20°C until analyzed using qPCR.An established protocol was used for extraction and purification of total RNA with a few modifications . The celnirK, nrfA, nosZ, and recA genes in the cDNA solutions were determined using the qPCR technique. nirK and nosZ genes were present in single copies in S. loihica strain PV-4 genome and only nrfA0844 of the two nrfA-like genes was targeted, as the expression of nrfA0505 did not correlate with respiratory ammonification activity  using SYBR Green detection chemistry. 2X Power SYBR Green PCR Master Mix Solution (Applied Biosystems) was used to prepare the reaction mix and a two temperature-cycle procedure was used for qPCR, with 95°C denaturation step (15 s) followed by 60°C elongation step (60 s) in each of 40 cycles. The amplification efficiencies ranged between 94.0 and 99.8% and the R2 values of the calibration curves were no less than 0.998 (Table 1). No amplification was observed in the negative controls without target DNA or cDNA. The qPCR assays for all four target genes were reliable down to 101 copies/μL, and the preparations with 1 copy/μL did not yield consistent results. Consistent melting curves confirmed the specificity of the qPCR reactions.The quantities of activity . The prisoftware  and a prre . The RT-qPCR reactions were performed with the samples collected from triplicate reaction vessels and independently processed through extraction, purification and reverse transcription procedures. Statistical analyses were performed with the data transformed to a logarithmic scale.S. loihica strain PV-4 reduced NO3– to NH4+ (respiratory ammonification) or to N2 via N2O (through denitrification) and also, whether the batch system functioned as a sink or a source of N2O during NO3– reduction . At all three pH conditions tested, all of NO3– added to a nominal concentration of 1.0 mM was consumed after 28 – 72 h after inoculation. The strain PV-4 culture incubated at pH 7.0 had the shortest lag period (∼8 h), while pH 6 and pH 8 cultures both had longer lag phases, as significant decreases in NO3– concentrations and increases in the cell densities were observed 35 and 22 h after inoculation, respectively. The maximum observed rates of NO3– reduction  and exponential growth rates  indicated that neutral pH was optimal for S. loihica strain PV-4, but also that overall cellular function of S. loihica strain PV-4 was not substantially compromised by the shift of pH within the examined range.pH conditions determined whether 2–accumulation. Accumulation of NO2– was not observed in the culture incubated at pH 6.0 and the concentration of NO2– remained below the detection limit throughout the experiment, indicating that potential NO2– reduction rate was at least as high as the rate of NO3– reduction. Contrastingly, at pH 7.0 and 8.0, NO2– accumulations up to 61.7 ± 2.6 μmoles/bottle and 82.6 ± 9.0 μmoles/bottle were observed, respectively, indicating that the rates of NO2– reduction were slower than the rates of NO3– reduction.The strain PV-4 cultures incubated at different pH conditions were distinguished by the magnitudes of transient NOS. loihica strain PV-4 functioned as a net sink of N2O, as N2O concentrations decreased consistently throughout incubation periods . No transient N2O accumulation was observed in the cultures incubated at pH 7.0 and 8.0. A clearly distinct trend was observed in the strain PV-4 culture incubated at pH 6.0, as a transient accumulation of N2O up to 72.3 ± 4.5 μmoles N2O-N/bottle  occurred before NO3– depletion. Although N2O accumulation was observed, N2O reduction was active throughout the experiment. The maximum N2O reduction rate of 12.2 μmoles N2O-N h-1 mL-1 OD600nm-1 was calculated at t = 54.5 h. This rate was comparable to the calculated maximum N2O reduction rate of 14.2 μmoles N2O-N h-1 mL-1 OD600nm-1 at pH 7.0 at t = 23.5 h. At pH 8.0, the maximum observed N2O reduction rate (279 nmoles N2O-N h-1mL-1 OD600nm-1 at t = 40 h) was substantially lower than those observed at pH 6.0 and 7.0. N2O reduction stopped after 60 h, when the dissolved N2O concentration was lowered to 0.75 μM and no other electron acceptor was available in the medium. Energy gained from N2O reduction may not be sufficient per se to support growth at the alkaline pH. In fact, no significant growth or N2O consumption was observed for 200 h when strain PV-4 was incubated with N2O as the sole electron acceptor at pH 8.0 (data not shown).At pH 7.0 and 8.0, 3– fate in the strain PV-4 cultures. Nitrogen mass balance was used to estimate the magnitude of denitrification activity, assuming that >90% of NO3–/NO2– was dissimilated to either NH4+ or denitrification products  in the amount of NH4+ was observed after t = 66.5 h. At pH 7.0, an increase in the NH4+ concentration was observed between t = 25 h and t = 27 h and ∼9.6% (9.7 ± 2.3 μmoles) of initially added 101.6 ± 2.1 μmoles NO3– was reduced to NH4+; however, NH4+ was still a minor product. The distribution of products from NO3– reduction at pH 8.0 was distinctively different from the results observed at pH 6.0 and 7.0. NH4+ was the major product of NO3– reduction, as 83.5 ± 1.2 μmoles of NO3– was reduced to NH4+. Respiratory ammonification dominated NO3– reduction at the alkaline pH and shutdown of the NirK-mediated NO2– reduction activity could be inferred from the mass balance. With diminished denitrification activity, S. loihica strain PV-4 cultures functioned as an N2O sink at pH 8.0 despite of reduced N2O reduction activity.pH was also a determinant of NOproducts . At pH 6S. loihica strain PV-4 cultures were amended with NO2– instead of NO3–, the effects of pH on NO2– reduction pathways were obscured by the effect of NO2– . At pH 6.0, neither cell growth or significant change in concentrations of NO2–, NH4+, or N2O was observed for >300 h. Lowering of the initial NO2– concentration to 0.1 mM did not result in cell growth or metabolic activity (data not shown), precluding the possibility that the toxicity of HNO2 at the acidic pH was the cause of growth inhibition. The experiments at pH 7.0 and pH 8.0 yielded statistically indistinguishable results (p > 0.05), as NH4+ was the predominant product  regardless of pH. No significant change in the concentration of N2O was observed at either pH, indicating that the pathways leading to the production and consumption of N2O were inactive when S. loihica strain PV-4 was incubated with NO2–. These results suggested that the pH effect on nitrogen-oxide dissimilation was eclipsed by the NO2– effect.When S. loihica strain PV-4 . To account for the differences in the cell densities and overall metabolic activities at different culturing conditions and growth stages, the nosZ transcription data were normalized with the recA transcription data. At pH 6.0 and pH 7.0, nirK transcription levels were at least five-fold higher than those of nrfA throughout the incubation periods except at t = 60 h at pH 6.0. The reduced transcription of nirK at this time point may be due to the depletion of NO3–. At pH 8.0, the differences in transcription of nrfA and nirK were insignificant (p > 0.05) throughout incubation . These transcription profiles explained the dominance of denitrification activity at pH 6.0 and 7.0 and the predominance of respiratory ammonification activity at pH 8.0. As expected from the sustained N2O reduction activity at pH 6.0, nosZ transcription was not adversely affected by the acidic pH . Transcription of nosZ was significantly more active at pH 6.0 and 7.0 than at pH 8.0, as the maximum nosZ transcripts / recA transcript values of 7.28 ± 2.59, 3.15 ± 1.50, and 0.62 ± 0.16 were recovered from samples extracted from the mid-exponential-phase cultures incubated at pH 6.0 (at t = 48 h), 7.0 (at t = 27 h), and 8.0 (at t = 52 h), respectively.Transcription analyses were performed using RT-qPCR technique with samples extracted at different time points during incubation of S. loihica strain PV-4 cultures grown with NO2– and N2O as the electron acceptors, distinguishable shifts in nirK, nrfA, and nosZ transcription levels from the lag phase (t = 17 h for pH 7.0 and t = 20 h for pH 8.0) to the mid-exponential phase  were observed . At the mid-exponential phase when active NO3–/NO2– reduction occurred, nrfA transcription levels were an order of magnitude higher than nirK transcription levels. The nirK transcription-to-nrfA transcription ratios were statistically indistinguishable between pH 7.0 and pH 8.0 (p > 0.05). These observations were in agreement with the predominance of respiratory ammonification. The diminished transcription of the genes responsible for N2O-producing reactions (nirK) and N2O-consuming reaction (nosZ) explained the absence of N2O production or consumption in the cultures amended with NO2– and N2O.In the samples extracted from S. loihica strain PV-4 with NO3– and N2O as electron acceptors confirmed the previous finding that pH is a significant environmental parameter that regulate nitrogen-oxide dissimilation reactions. pH was previously suggested as one of the environmental parameters that determine the fate of NO3– in axenic cultures of S. loihica strain PV-4 and also, in complex mixed cultures ; however, toxicity of NO2– at 1.0 mM concentration was not sufficient to have an adverse impact on cell growth (2) is known to be more toxic than NO2– . Thus, the absence of growth with 0.1 mM NO2– suggested that the HNO2 toxicity was not the cause for the growth inhibition at pH 6.0. Although largely speculative, the absence of growth may be explained with the differential transcription levels and activities of the enzymes involved with nitrogen-oxide dissimilation at the varying pH conditions. The elevated NO2–:NO3– ratio could have resulted in down-regulation of nirK and nosZ transcription and up-regulation of nrfA transcription. As NrfA has diminished activity at low pH, S. loihica strain PV-4 may lack active NO2–-reducing enzymes and thus, may not be able to generate sufficient energy for growth.The absence of growth with NOl growth . Nitroushan NO2– . As HNO22O accumulation observed during NO3– reduction by S. loihica strain PV-4 was consistent with the observations made previously with P. denitrificans and soil microbial consortia  under acidic pH was previously observed upon incubation of soil inoculum  as relative to N2O-removal reaction  may be one of the major cause of enhanced N2O emissions from moderately acidic environments.The transient Nonsortia . N2O peamulation . Insteadinoculum . Our obs3–/NO2– reduction pathway even under alkaline conditions (S. loihica strain PV-4 was previously observed to be hierarchical, as the effect of C:N ratio overshadowed the effect of NO2–:NO3– ratio when either lactate or NO3– was limiting (2–:NO3– effect in this study, as NO2– was reduced exclusively via respiratory ammonification pathway regardless of pH. This hierarchical regulation may be applicable to soils and sediments and pH may be a determinant of the fate of NO3– and NO2– only when their reduction pathway is not predetermined by overarching environmental factors. Agricultural soils simultaneously exhibiting both denitrification and DNRA activities are not rare (2O. Pure culture experiments may not be sufficient to portray the complex nature of environmental systems; however, the observations with S. loihica strain PV-4 certainly demonstrate the feasibility of manipulation of soil nitrogen cycling to simultaneously reduce N2O emission and promote N retention via pH control.The positive correlation of DNRA activity with pH has been observed in experiments with soils and sediments , althougnditions , 2014. Rlimiting . Likewisnot rare . pH contHK performed the experiments and analyzed data. HK and DP developed the experimental methodology for RT-qPCR assays. SY planned the research and designed the experiments. HK and SY wrote the manuscript. All authors discussed results and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It displays superior ablation resistance at temperatures from 2,000–3,000 °C, compared to existing ultra-high temperature ceramics . The carbide is a substitutional solid solution of Zr–Ti containing carbon vacancies that are randomly occupied by boron atoms. The sealing ability of the ceramic’s oxides, slow oxygen diffusion and a dense and gradient distribution of ceramic result in much slower loss of protective oxide layers formed during ablation than other ceramic systems, leading to the superior ablation resistance.Ultra-high temperature ceramics are desirable for applications in the hypersonic vehicle, rockets, re-entry spacecraft and defence sectors, but few materials can currently satisfy the associated high temperature ablation requirements. Here we design and fabricate a carbide (Zr Hypersonic and aerospace applications motivate development of materials with improved resistance against ablation and oxidation at high temperatures. Here authors demonstrate a quaternary carbide, where sealing by surface oxides, slow oxygen diffusion and a graded structure yield improved ablation resistance over established ceramics. Currently, one of the biggest challenges is how to protect critical components such as leading edges, combustors and nose tips so that they survive the severe oxidation and extreme scouring of heat fluxes at temperatures in excess of 2,000 °C during flight132 ablation resistance, much attention has been focused on adding silicides  making it a poor option for anti-ablation applications171820To reduce the evaporation of boron oxides and to improve ZrB122 ref.  and so o122 ref.  to ZrB2 0.8-Ti0.2-C0.74-B0.26, laid down on a C/C composite by reactive melt infiltration (RMI) and pack cementation (PC) is proposed  and linear ablation rate (LAR) characterize the mass loss and dimensional stability of the materials, respectively. In general, a high MAR  and LAR  indicate poor ablation performance. Hence, the MAR and LAR results for our carbide in 2–SiC ceramics fabricated by spark plasma sintering (SPS). For instance, the LAR of ZrC at 2,500 °C is 8.0 μm s−1 and MAR is 1.10 mg cm−2 s−1, whereas our carbide gains 3.5 μm s−1 in thickness and 0.14 mg cm−2 s−1 in weight. This is because the oxide layer expands and increases the weight countering any material loss from ablation, indicating an almost negligible loss of our carbide. This is over 12 times better than the loss of material for ZrC (assuming both carbides have the same volume expansion of oxides per second). It is noteworthy that the MARs of our carbide from 60–120 s at 2,000–2,500 °C distribute around the zero, indicating a slight weigh loss or weigh gain. These higher negative values of LARs obviously distinguish it from other UHTCs indicated by the green arrows in 0.8Ti0.2C fabricated by RMI), and typical of the ablation flame of ZrB2 (ref. 2) show better levels of oxidation resistance than their carbides 0.8Ti0.2C0.74B0.26 and Zr0.8Ti0.2C shown in 0.8Ti0.2C. In addition, as shown in 0.8Ti0.2C0.74B0.26 carbide exhibits a level of ablation resistance and protection not seen in other Zr-based carbides and diborides and common high-temperature composites. The results also suggest that C/C composite modified by Zr0.8Ti0.2C0.74B0.26 , and the pores below the surface into the test-piece. The surface region comprises up to 75% ceramic and 25% C. It is sufficiently dense that the top surface  as shown in 0.8Ti0.2Cx)(1− (0<x<1) (Methods section and 0.8Ti0.2C0.74B0.26 forms through the boration of Zr0.8Ti0.2Cx)(1- via solid diffusion of boron atoms (see following reactions) during PC process. Zr0.8Ti0.2Cx)(1− have been built by disordered carbon vacancies (possibly having short-range-order0.8Ti0.2Cx), 0.8Ti0.2Cx) governs the main reaction of boron atoms. 2O3 may react with carbon fibres and pyrocarbon, as shown in reaction (3), which is another source of the B4C in reaction (1). Reaction thermodynamics are referenced in where 0.8Ti0.2C0.74B0.26 and SiC are oxidized and converted into Zr0.80T0.20O2, B2O3 and SiO2, respectively, as shown in 0.80Ti0.20O2 partially melts and forms a relatively dense layer in the central ablated surface. However, the evaporants with low melting point, such as SiO2 and B2O3, escape from the oxide layer leaving evacuation channels: the holes, as shown in 0.80Ti0.20O2 crystals connected by the melt become larger and the holes evidently shrink as shown in 0.80Ti0.20O2, zirconium titanate and SiO2, is formed as shown in 0.80T0.20O2 layer has effectively retained the boron and silicon and extended their consumption time, according to their residual contents shown in 0.80Ti0.20O2 displays a durable and robust structure comprising grain skeletons and liquid phases as shown in 0.80Ti0.20O2 during the ablation test and decreased the viscosity of pure ZrO2 melt due to the lower melting point of TiO2  than that of ZrO2 , which conferred a self-healing ability on the oxide layer, instead of a porous layer. Meanwhile this evidently decreased the vaporization and loss rate of oxides, compared with the conventional ZrB2–SiC or ZrC–SiC ceramic systems. For instance, the vaporization rate (VR) of TiO2 is 0.23 mm s−1 at 2,227 °C, whereas the VR of SiO2 (207 mm s−1) is around 900 times higher than that of TiO2 at the same temperature0.8Ti0.2C0.74B0.26 will result in much less loss of oxide because of vaporization and good adhesion to the substrate shown in 2-SiO2 system from the oxidation of ZrB2-SiC and ZrC-SiC. Second, the good ablation response is due to the low oxygen permeability (OP) of the oxide layer. In this work, the Zr0.80Ti0.20O2 layer on the quaternary carbide has effectively caught the B2O3 and SiO2 which has a very low OP 0.8Ti0.2C0.74B0.26 at different ablation temperatures. Moreover, it is believed that the intrinsic oxygen diffusion coefficient (ODC) of the Zr0.80T0.20O2 layer is lower than that of the pure ZrO2 due to the lower ODC of TiO2  compared with ZrO2 0.80Ti0.20O2 leads to less formation and loss of oxides, and further improves the ablation resistance of carbide, though the melting point of oxide (Zr0.80Ti0.20O2) is lower than 3,000 °C. Consequently, it is believed that our carbide displaying a better ablation resistance than the conventional ceramics can increase the survival time of the components in extremely oxidizing environments up to 3,000 °C. In addition, the lower boron content in Zr0.8Ti0.2C0.74B0.26 compared with that in ZrB2 leads to a lower mass loss and fewer defects such as pores and cracks originating from the evacuation channels of B2O3 at higher temperature, which is significantly beneficial to the ablation response. Third, it also benefits from the gradual transition from ceramic to carbon composite. A dense and high volume of ceramic is more conducive to forming a dense oxide layer and ensured the minimum damage to the underlying carbon matrix from the extreme hot and oxidizing gas.The carbide presented here exhibits superior ablation response compared to current UHTCs. This can be attributed to the following facts. First, a relatively dense oxide layer with a suitable viscosity plays a key role in resisting the ablation of extremely hot gas. Generally, loose scale with a very high viscosity , or a liquid layer with a very low viscosity , provides poor protection because of the tendency for detaching or splashing of oxidesxBy), where A and M are the main atoms  and the substitution atoms , respectively. For instance, Hfx (Zry1/Tiy2/Tay3)C0.8B0.2, Zrx (Tiy1/Tay2)C0.8B0.2 and TiC0.8B0.2 and so on, can be built, according to similar methods. To increase ablation resistance across a wide range of temperatures, a carbide’s oxides layer that owns low VR, OP, good self-healing ability and adhesion strength to the substrate can be achieved through the doping contents of different substitution atoms (yi) of ceramic with a variety of melting points. Consequently, we develop an effective means of fabricating ablation resistant UHTCs. Moreover, these ceramics can be fabricated into powders, bulk materials and layers to extend their application. For instance, in addition to the potential use in hypersonic vehicles, it is expected that they can be used as the nozzle throats and diffusers for reusable rockets, which requires a very low ablation rate to extend the lifetime for recyclability at low cost. Other potential uses may include the hot section components in re-entry spacecraft, defence army, gas turbines and nuclear areas and so on.In conclusion, we have designed a carbide assembled by solid solution and atomic diffusion during PC and RMI. Importantly, the ceramic layer displays better ablation resistance  under oxidizing environments up to 3,000 °C relative to existing common UHTCs and high temperature composites. More broadly, this work provides a platform for building a series of such UHTCs  C−3 density using pyrocarbon deposited by CVI using CH4 and H2 gases at 900–1,000 °C. The open porosity of the composites with NIP ranged from 39.8 % to 28.8 %. For instance, a sample having a density of 1.16 g cm−3 and an open porosity of 34.3 % possessed a modal pore diameter of 42.0 μm shown in 0.8Ti0.2Cx) and mass ablation rate (MAR) were calculated according to t is the ablating time. S is the ablation area. Ablation rate is averaged over three specimens. The tests lasted 60 and 120 s.The ablation behaviours of the samples were evaluated using an oxyacetylene flame . During θ scan range was 10–90°, scanning resolution was 0.05° per step. For the measurement of crystals structure, the samples were scanned between 10–90° 2θ, at a step-width of 0.02° and scan speed of 0.5°·per min. Rietveld refinement was carried out with the program Maud using a pseudo-Voigt profile function383D X-ray computed tomography (CT) was conducted using a Zeiss Xradia Versa 520 X-ray microscope at the Henry Moseley X-ray Imaging Facility . The accelerating voltage and current of the X-ray tube were set as 140 kV and 72 mA, respectively. Each scan comprised 1,601 radiographs taken incrementally over a rotation angle of 360˚. A 3D volume rendering of the sample was created from the virtual slices in AVIZO software. The first 100 slices and last 100 slices were cropped due to low image quality presumably as a result of the cone-beam geometry. Pores, carbon and ceramics were segmented out using the top hat methodA super probe electron probe microanalysis system  was used to detect the content and distribution of elements. High resolution transmission electron microscope (HRTEM) images and selected area electron diffraction (SAED) patterns were obtained with an FEI Talos F200A microscope equipped with an X-FEG electron source. TEM samples were prepared by focused ion beam  using the in-situ lift-out technique on cross-sections of the samplesThe authors declare that the data supporting the findings of this study are available from the corresponding authors on reasonable request.How to cite this article: Zeng, Y. et al. Ablation-resistant carbide Zr0.8Ti0.2C0.74B0.26 for oxidizing environments up to 3,000 °C. Nat. Commun.8, 15836 doi: 10.1038/ncomms15836 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary ReferencesVideo of 3D X-ray tomography of compositesVideo of 120 s ablation test at 2000 °C.


N-substituted maleimide in stereoselective fashion.The one-pot [3 + 2] cycloaddition of an azomethine ylide with a maleimide followed by another [3 + 2] cycloaddition of an azide with the second maleimide gives a 1,5-diamino intermediate which is used for a sequential aminomethylation reaction with formaldehyde through [5 + 1] annulation to afford a novel polycyclic scaffold bearing tetrahydroquinazoline, pyrrolidine, pyrrolidinedione, and  I–VI bearing heterocyclic rings such as hydantoin, pyrrolidine, pyrrolidinedione, piperazinedione, and dihydrobenzodiazepinedione  reactions is an attractive green organic technique –5. By thinedione   annulation reactions. But only formaldehyde afforded tetrahydroquinazoline 1a in good yield under catalyst-free conditions. A number of [5 + 1] annulation reactions using selected compounds 7 were carried out to afford 10 analogs of tetrahydroquinazolines 1 in 88–95% isolated yields as single diastereomers  and 13C NMR spectra (75 or 101 MHz) were recorded on Agilent NMR spectrometers. Chemical shifts were reported in parts per million (ppm), and the residual solvent peak was used as an internal reference: proton , carbon (chloroform δ 77.0). Multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet), br s (broad singlet). Coupling constants were reported in hertz (Hz). LC–MS was performed on an Agilent 2100 LC with a 6130 quadrupole MS spectrometer. A C18 column  was employed for the separation. The mobile phases were MeOH and H2O both of which contained 0.05% CF3CO2H. A linear gradient from 25:75 (v/v) MeOH/water to 100% MeOH over 7.0 min at a flow rate of 0.7 mL/min was employed as a mobile phase. UV detections were conducted at 210 nm, 254 nm and 365 nm. Low resolution mass spectra were recorded with APCI . The final products were purified on Angela HP-100 pre-LC system with a Venusil PrepG C18 column .Chemicals and solvents were purchased from commercial suppliers and used as received. 3 (1.2 mmol), 2-azidobenzaldehyde , and maleimide 5 (1.0 mmol) in 2.0 mL of CH3CN was added Et3N (2.0 mmol). After being stirred at 25 °C for 5 min, the reaction mixture was heated by microwave irradiation at 115 °C for 25 min. Upon the completion of the reaction as monitored by LC–MS, maleimide 6 (1.0 mmol) was added to the reaction mixture and then heated by microwave irradiation at 125 °C for 25 min. The concentrated reaction mixture was isolated on a semi-preparative HPLC with a C18 column to afford purified product 7 as a single diastereomer.The following procedure is analogous to one of our previous procedures . To a so7 (0.5 mmol), in 1.0 mL of 1,4-dioxane was added formaldehyde solution . The reaction mixture was heated at 110 °C for 3 h. Upon the completion of the reaction as monitored by LC–MS, the reaction mixture was concentrated and then isolated on a semi-preparative HPLC with a C18 column to afford purified product 1.To a solution of compound File 1Compound characterization data, X-ray report, and copies of NMR spectra.