Ericaceae are common in arctic and alpine regions. Many of these plants are associated with ericoid mycorrhizal (ERM) fungi, which allow them to take nutrients and water from the soil under harsh environmental conditions and, thus, affect host plant survival. Despite the importance of ERM fungi to alpine plant communities, limited information is available on the effects of microhabitat and host identity on ERM fungal communities. We investigated the communities of putative ERM fungi isolated from five dwarf shrub species  that co-occur in an alpine region of Japan, with reference to distinct microhabitats provided by large stone pine (Pinus pumila) shrubs . We obtained 703 fungal isolates from 222 individual plants. These isolates were classified into 55 operational taxonomic units (OTUs) based on the sequencing of internal transcribed spacer regions in ribosomal DNA. These putative ERM fungal communities were dominated by Helotiales fungi for all host species. Cistella and Trimmatostroma species, which have rarely been detected in ERM roots in previous studies, were abundant. ERM fungal communities were significantly different among microhabitats (R2=0.28), while the host effect explained less variance in the fungal communities after excluding the microhabitat effect (R2=0.17). Our results suggest that the host effect on ERM fungal communities is minor and the distributions of hosts and fungal communities may be assessed based on microhabitat conditions.Dwarf shrubs of the family  Ericaceae and Diapensiaceae (e.g. Schizocodon and Diapensia)  is a symbiotic association between fungi and the roots of plants in the families apensia) , 53, 57.apensia) , 39, 59.apensia) . ERM hosapensia) , 34, 52. regions , 47, 59.Helotiales is the most dominant group (Rhizoscyphus ericae (=Hymenoscyphus ericae) and Oidiodendron species have frequently been isolated from ERM roots and have been confirmed as ERM-forming mycobionts in inoculation experiments  have recently been identified and sequenced  shrubs and ericaceous dwarf shrubs. Strong winds lead to desiccation in these habitats, which makes plant establishment difficult, leaving large areas of bare ground. However, once Japanese stone pine or Ericaceae plants are established, their shrubs alleviate the harsh environmental conditions locally and facilitate further seedling establishment, as reported by Perkins  co-existing in an alpine ecosystem. The hypotheses examined in this study are as follows: putative ERM fungal communities are affected more by microhabitats than by hosts, and host effects on putative ERM fungal communities become evident after excluding microhabitat effects.In the present study, we investigated putative ERM fungal communities with reference to microhabitat and host identity using five dwarf shrub species (Three study plots (0.5 ha each) were established on the exposed mountain ridges of Mt. Norikura, Kikyougahara , Daikokudake , and Fujimidake .Ericaceae species, i.e. A. nana, D. lapponica, E. nigrum, L. procumbens, and V. vitis-idaea, were found in all study plots and were used in this study. ERM formation in these plant species has already been reported  in January and the highest (12.0°C) in August, and mean annual precipitation is 2,738 mm, according to observations by the Japan Meteorological Agency . The site is typically covered by snow packs until late June . Five reported , 53, 57.2) were collected from four microhabitat categories: pine shrub (the center of P. pumila shrubs under dense shade from pine leaves), the east edge , the west edge (the western and windward edge of P. pumila shrubs), and open (the bare ground away from P. pumila shrubs). In the pine shrub habitat, we only collected V. vitis-idaea because no other Ericaceae plants were found. All five plant species were collected in the east edge, west edge, and open habitats. Individual plants were excavated with the surrounding soil, placed separately in plastic bags, and sealed and stored at 4°C during transport to the laboratory. A total of 26, 72, 76, and 48 individuals were collected in the pine shrub, east edge, west edge, and open habitats, respectively. Of the 222 plants, 60, 12, 48, 22, and 80 were A. nana, D. lapponica, E. nigrum, L. procumbens, and V. vitis-idaea, respectively. The number of plant samples collected for each species/microhabitat combination is listed in In early August 2014, mature plant samples  based on ≥97% similarity  using ATet al.  to test for significant differences in fungal communities among habitat categories and among hosts. Singletons and doubletons were excluded from community data matrices. These statistical analyses were performed using R with 9,999 permutations, applying the Bray–Curtis distance as a community dissimilarity index.The occurrence of an OTU was quantified as the number of individual plants isolated from that OTU. The biased occurrence of OTUs was tested using a weighted chi-squared test, in which the number of individuals collected was used as the expected occurrence. Chao2 values, which is a species richness estimator based on the occurrence of rare taxa, were calculated for each host and microhabitat category with sample-based rarefaction analyses using Estimate S var. 8.20  with 1,0The identified sequences were deposited at DDBJ under the accession numbers KY522913–KY522967.Capnodiales and one to Chaetothyriales. All remaining fungi belonged to the order Helotiales, apart from two OTUs, which were assigned only at the class level. Phialocephala fortinii sp. 1 had the most frequent occurrence and was found in 54 plants, followed by Cistella sp., found in 47 . NMDS ordination indicated that putative ERM fungal communities were clearly separated based on microhabitat , which was found to be significant in an Adonis test   in pineshrub, east edge, west edge, and open habitats were 20 (37.3), 29 (39.9), 37 (74.4), and 16 (25.1), respectively. No significant differences were found in putative ERM fungal communities between the east edge and west edge habitats  .A. nana, D. lapponica, E. nigrum, L. procumbens, and V. vitis-idaea were 27 (42.3), 4 (4.0), 21 (41.5), 9 (10.9), and 42 (67.9), respectively. Each of the hosts was uniquely associated with 6, 1, 5, 2, and 15 OTUs, respectively  were found in all hosts. Differences in occurrence between hosts were found in seven OTUs   on ectively . Two OTUa sp. 1) .p<0.001). However, the host effect was confounded by that of microhabitat because some hosts preferred a certain microhabitat. Nevertheless, even for an Adonis test with the effects of microhabitats excluded, the host effect was significant (p=0.001), but explained less variance in the community data (R2=0.17). When only edge habitat data were used, the fungal communities of E. nigrum were distinct from those of A. nana and V. vitis-idaea (p<0.001). The fungal communities of A. nana and V. vitis-idaea were tightly clustered in the NMDS plot (p=0.800). When fungal communities were compared among hosts in just the open habitat samples, the communities in D. lapponica were distinct from those in other hosts , while no significant difference was found among A. nana, E. nigrum, and L. procumbens.Overall, the host effect was significant in an Adonis test that did not exclude the effect of microhabitat , supporting our first hypothesis that habitat has an effect on putative ERM fungal communities. Ishida and Nordin , but was confounded by the effect of microhabitats because some host species showed a habitat preference. For example, V. vitis-idaea was the only species that was found under pine shrubs, while D. lapponica was mostly located in open habitats. Previous findings on potential host effects on ERM communities have been inconsistent, being significantly different in Bougoure et al.  and Cistella species in ERM roots. Capnodialean species have been reported to be the root-associated fungi of arctic plants, including ERM hosts, but at low frequencies (Cistella species are typically identified as soil fungi (Another interesting result is the dominance of quencies , 59. Capquencies , 21 and quencies , 21, 28.il fungi  and leafil fungi . AlthougAlpine dwarf shrubs of ericaceous plants under similar environmental conditions shared the majority of putative ERM fungi. This result indicates that most hosts are associated with generalist ERM fungi, which may, in turn, facilitate seedling establishment in a broad range of hosts and allow these hosts to co-exist. This facilitation is often reported in ectomycorrhizal systems, but not in ERM associations , 38, 41.


LOXL1/LOXL2 gene expression and protein levels were increased in IPF versus non-IPF. Increased collagen fibril thickness in IPF versus non-IPF lung tissues correlated with increased LOXL1/LOXL2, and decreased LOX, protein expression. β-Aminoproprionitrile  but not Compound A (LOXL2-specific inhibitor) interfered with transforming growth factor-β-induced collagen remodelling in both models. The β-APN treatment group was tested further, and β-APN was found to interfere with stiffening in the decellularized matrix model. LOXL1 activity might drive collagen remodelling in IPF lungs. The interrelationship between collagen structural remodelling and LOs is disrupted in IPF lungs. Inhibition of LO activity alleviates fibrosis by limiting fibrillar collagen cross-linking, thereby potentially impeding the formation of a pathological microenvironment in IPF.Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with few effective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linking mediated by the lysyl oxidase (LO) family of enzymes ] might contribute to disease pathogenesis and represent a therapeutic target. This study aimed to further our understanding of the mechanisms by which LO inhibitors might improve lung fibrosis. Lung tissues from IPF and non-IPF subjects were examined for collagen structure (second harmonic generation imaging) and LO gene  and protein (immunohistochemistry and western blotting) levels. Functional effects (collagen structure and tissue stiffness using atomic force microscopy) of LO inhibitors on collagen remodelling were examined in two models, collagen hydrogels and decellularized human lung matrices.  Summary: Transforming growth factor-β-induced collagen remodelling is driven by enhanced lysyl oxidase expression in idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive fibrosing lung disease of unknown cause that leads to chronic respiratory failure and death , with a Like many fibrotic disorders, IPF is characterized by enhanced deposition and remodelling of the extracellular matrix (ECM). Although data are emerging about the altered composition of the ECM in IPF , less isin vitro, ex vivo and in vivo (Fibrillar collagen structures (predominantly collagens I and III) can be visualized and quantified using second harmonic generation (SHG) microscopy . SHG ima in vivo  and can  in vivo . This te in vivo  and even in vivo . We have in vivo .Maturity of collagen fibres and thus the mechanical properties of the fibres are dependent on the level of intermolecular cross-linking. Although the intermolecular fibrillar organization and certain cross-linking processes can happen spontaneously, enzymes such as lysyl oxidases (LOs) are necessary for further cross-linking. The LO family of enzymes consists of five known paralogues: lysyl oxidase (LOX) and LOX like 1-4 (LOXL1-4). During normal development, the expression of LOs is tightly regulated, with aberrant expression and activity being associated with pathological manifestation in various diseases, including cancer  and, as ClinicalTrials.gov NCT01769196 .To investigate the collagen structural remodelling in IPF, SHG microscopy was performed on lung tissue sections from non-IPF and IPF subjects. The SHG forward (F) signal is predominantly from mature/organized fibrillar collagen, whereas the backward (B) signal is from immature/disorganized fibrillar collagen. Thus, increased F/B area corresponds to increased amounts of mature/immature collagen, whereas increased F/B intensity ratios correspond to an increased degree of collagen maturity/thickness and vice versa . QuantifLOXL1 gene expression was increased in IPF lung tissue compared with healthy controls in two independent studies [false discovery rate (FDR) adjusted P-value <0.25], whereas the LOXL2 gene expression was increased in IPF lung tissue in a single data set. No differences were found in LOX gene expression levels.Fibrillar collagen maturity is catalysed by the LO family of enzymes. To investigate the levels of LO enzymes in lung tissues, lung tissue gene expression profiles of IPF and healthy controls were analysed from two publically available data sets . LOXL1 gTable S1). In vitro, primary human lung parenchymal fibroblasts from IPF patients produced significantly greater levels of LOXL2 but not LOX or LOXL1 in the cell lysate compared with non-IPF controls .To confirm the findings from the gene expression study, tissue sections from the same samples used for the examination of collagen structural organization were examined for LOX, LOXL1 and LOXL2 protein levels. Quantification of the captured images of the stained lung tissue sections showed that LOX percentage surface area and density were decreased in IPF compared with non-IPF, whereas the percentage surface area and density of LOXL1 was increased in IPF compared with non-IPF tissues A. In agrFig. S3) and LOXL2  densities and LOX/LOXL1 percentage surface areas were significantly decreased in IPF lung tissue samples compared with non-IPF subjects . The ratio of LOX/LOXL1 percentage surface area inversely correlated with collagen maturity/organization .The ratio of LOX/LOXL1 B and LOXion F/B; C. There Table S2 shows the strength of the contribution of the variables  to the two relationship groups. The strengths of contributions of the variables were used to determine the weighted average values for each relationship group . The pan-inhibitor of LOs, β-APN, returned TGF-β-induced collagen remodelling to baseline. In addition, the LOXL2-specific inhibitor blocked TGF-β-induced remodelling of fibrillar collagen; remodelling levels in the presence of either inhibitor without TGF-β were not different compared with control  was measured by atomic force microscopy . TGF-β induced a significant increase in tissue stiffness that was inhibited by the pan-inhibitor of LOs, β-APN . IndividLOXL1 and LOXL2 gene and protein levels are increased in the lungs of IPF compared with non-IPF subjects, with a positive correlation between increased protein expression and increased fibrillar collagen organization.Although increased collagen deposition is well appreciated in fibrotic lung tissues, this study identifies, for the first time, alterations in the fibrillar organization of the collagen fibres in IPF lung tissue. In addition, this study reports that ClinicalTrials.gov NCT01769196) was halted early because of a lack of efficacy  compared with non-diseased subjects. It can be postulated that the increased collagen fibre maturity/thickness contributes to the increased stiffness characteristic of IPF lungs . This isLOXL1 gene and protein expression in IPF lungs, compared with non-IPF tissues, was observed, with a positive correlation of protein levels with collagen organization. By contrast, LOX gene expression was unchanged, whereas the LOX protein level was decreased in IPF lung, highlighting differential LO family expression in IPF lung tissue, which is similar to that reported during tumour progression and metastasis  culture express a 50 kDa proteolytically processed form of LOXL2 , rather Fig. S7), but the present experiment was not designed to address this hypothesis.It is worth noting that the stiffness measured in the reseeded decellularized matrices (mean 40-60 kPa) was higher than the values measured in native human lung parenchymal tissue . After formaldehyde fixation, isolated lung tissues were embedded in paraffin.Tissue isolation was performed on fresh explanted lung tissues. Human lung parenchymal tissues were isolated and cut into pieces no bigger than 1 cmPrimary human lung fibroblasts were isolated as previously described  and were2 incubator (5% CO2 in air) at 37°C. Growth media were aspirated from cells every 4 days and replaced with fresh growth media until confluence.Primary human lung fibroblasts were grown in growth media [DMEM low glucose (Gibco) with 0.025 M HEPES (Gibco) and 0.375% sodium hydrogen carbonate ] incubated in a humidified CO3VO4; Sigma), 1% (v/v) Triton X-100 (Sigma), 10% (v/v) glycerol (Sigma), 0.1% (w/v) SDS (Amresco), 0.5% (w/v) sodium deoxycholate (Sigma), 1 mM PMSF , 1% (v/v) Protease Inhibitor Cocktail Set III ] on ice, scraped into a tube, and centrifuged at 150×g for 5 min. The supernatants were collected and stored at −80°C for further experiments. Total sample protein was measured using a standard BCA assay (Sigma).Whole-cell lysates were prepared using protein extraction buffer  containing resuspended cells was then added at higher cell concentration for dilution in the collagen gel solution . Collagen gel mixed with cells  was pipetted into a 48-well plate and allowed to set in a humidified CO2 incubator at 37°C for 2 h. Once the gel had set, the gels were then released from the wells by gently running a sterile syringe needle around between the gel and the wall of the wells. Treatment media  of the same volume as the gel were pipetted gently on top of the gels in the wells. The collagen gels were collected at day 7, fixed in 4% paraformaldehyde solution and paraffin embedded.Collagen gel solution was prepared for a final collagen gel concentration of 2 mg mlDecellularized tissues from a non-diseased subject and an IPF subject were prepared as previously described . Essenti5 fibroblasts ml−1 in growth media at a ratio of 1 matrix:2 ml of cell solution  up to a maximum of 25 ml of solution in a 50 ml Falcon tube. The Falcon tubes containing the matrices and cell suspension were mounted on a suspension mixer  and constantly rotated at low speed (approximately one rotation every 20 s) at 37°C for 24 h. The reseeded ECMs were placed in separate wells of a 48-well plate with fresh media [0.01% DMSO +0.1% BSA ±10 ng ml−1 TGF-β  ±300 nM Compound A or 0.1 mM β-APN (Sigma)] and incubated at 37°C and 5% CO2. The reseeded decellularized matrices were collected at day 7; half were fixed in 4% paraformaldehyde solution for 24 h and paraffin embedded, whereas the other half were fresh frozen in optimal cutting temperature (OCT) compound and stored at −80°C.Decellularized lung slices from both non-diseased and IPF subjects were washed three times in sterile DMEM solution and were subsequently added to a suspension of 5.0×10AFM measurements were performed on decellularized human lung matrices with or without reseeded non-IPF cells. Matrices were embedded in optimal OCT compound, and 10-µm-thick tissue slices were cryosectioned at −21°C and mounted on poly-L-lysine-coated glass slides. PBS solution was added on the tissue slice to avoid drying.−1 by the thermal fluctuation method .Parenchymal tissue areas were identified under an optical microscope  and mechanically analysed with a BioScope Catalyst AFM . Microindentations were performed using a 2.5-µm-radius sphere-tipped probe  with a spring constant determined at ∼100 pN nmn method . For eacE, was determined by fitting of the force curve by the Hertz sphere model  or the Mann–Whitney U-test  as appropriate. Kruskal–Wallis and two-way ANOVA statistical analyses were performed where appropriate. Factor analysis was performed using SPSS software  using ‘principal axis factoring’ analysing ‘correlation matrix’ and extracting ‘2’ factors from the data set. Promax rotation was used for the factor analysis, using a regression method and excluding cases listwise where there were missing data points.Data were entered and sorted using Microsoft Excel . Sample sizes for comparing IPF versus non-IPF were determined based on a previous study . Statist


A 4 × 5 design was utilized, with four composites , and five water-ageing time periods . After each water-ageing period, the mechanical properties of the resins were measured in a three-point flexure, and antibacterial properties were tested via a dental plaque biofilm model using human saliva as an inoculum. After 12 months of water-ageing, NACP-QADM-NAg had a flexural strength and elastic modulus matching those of the commercial control (P>0.1). Incorporation of QADM or NAg into the NACP composite greatly reduced biofilm viability, metabolic activity and acid production. A composite containing both QADM and NAg possessed a stronger antibacterial capability than one with QADM or NAg alone (P<0.05). The anti-biofilm activity was maintained after 12 months of water-ageing and showed no significant decrease with increasing time (P>0.1). In conclusion, the NACP-QADM-NAg composite decreased biofilm viability and lactic acid production, while matching the load-bearing capability of a commercial composite. There was no decrease in its antibacterial properties after 1 year of water-ageing. The durable antibacterial and mechanical properties indicate that NACP-QADM-NAg composites may be useful in dental restorations to combat caries.Dental composites are commonly used restorative materials; however, secondary caries due to biofilm acids remains a major problem. The objectives of this study were (1) to develop a composite containing quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP), and (2) to conduct the first investigation of the mechanical properties, biofilm response and acid production  This approach enables a high antibacterial activity at a low Ag filler level, without compromising the composite colour and mechanical properties.33Dental caries is a prevalent worldwide problem,38 CaP biomaterials are important because of their excellent biocompatibility, bioactivity, and similarity to minerals in hard tissues.42 Composites containing nanoparticles of amorphous calcium phosphate (NACP) release calcium (Ca) and phosphate (P) ions similar to traditional CaP composites, but possess much better mechanical properties.43 NACP composites are “smart” and greatly increase the release of Ca and P ions at an acidic pH, at which these ions would be most needed to inhibit caries.42 In addition, NACP composites have achieved enamel lesion remineralization rates fourfold higher than that of a fluoride-releasing commercial dental composite.38Another approach to combat caries has been the development of calcium phosphate (CaP)-containing resins.43 However, long-term experiments are still needed to investigate the antibacterial durability of the dental resins. A previous study has shown that a composite containing MDPB has a strong antibacterial effect after 3 months of immersion in water.19 In another study, the antibacterial activity of a composite containing PEI nanoparticles has been found to be maintained after 1 month of water-ageing.25 In addition, the antimicrobial activity of a QAM-containing acrylic resin has been shown to be maintained after 3 months of water-ageing.44 However, whether the antibacterial effects decrease after 6 months is unclear, and longer term studies  have not been reported for antibacterial dental resins.To further enhance the anti-caries efficacy, the antibacterial approach and the CaP approach have been combined in the same resin, so that the antibacterial agent can reduce biofilms while the Ca and P ions remineralize lesions and inhibit caries.Accordingly, the objectives of this study were to develop a bioactive composite containing QADM, NAg, and NACP for the inhibition of caries, and to conduct the first investigation of the mechanical properties, oral biofilm response and lactic acid production as a function of water-ageing time from 1 day to 12 months. We hypothesized that: (1) an NACP composite containing QADM and NAg would have much less biofilm viability and acid production than a commercial composite control; (2) the antibacterial activity of the NACP-QADM-NAg composite would show no significant decrease with increasing water-ageing time from 1 day to 1 year; and (3) the NACP-QADM-NAg composite would possess similar mechanical properties to those of a commercial control composite after a long-term water-ageing treatment.3[PO4]2) were synthesized via a spray-drying technique, as described in a previous study.42 Briefly, calcium carbonate  and anhydrous dicalcium phosphate  were dissolved in acetic acid to obtain Ca and P ion concentrations of 8 mmol·L−1 and 5.333 mmol·L−1; respectively, yielding a Ca/P molar ratio of 1.5. This solution was sprayed into the heated chamber of a spray-drying apparatus, and an electrostatic precipitator was used to collect the dried particles. This process produced NACP with a mean particle size of ~116 nm.42 NACP consisted of individual particles as well as some clusters.43 The clusters contained numerous small particles that were probably stuck together in the spray-drying chamber before the particles were completely dried. Whereas the individual particles had sizes on the order of 10 nm, the clusters had sizes of ~100–300 nm.43 Measurement of 100 random particles in a previous study has shown an average size of 37 nm for the individual particles, and an average size of 225 nm for the clusters.43NACP  and triethylene glycol dimethacrylate (TEGDMA) at 1:1 mass ratio was rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-32 The bis(2-methacryloyloxyethyl) dimethylammonium bromide was a dimethacrylate, which has been identified as QADM in previous studies.32 Briefly, 10 mmol of 2-ethyl methacrylate  and 10 mmol of 2-bromoethyl methacrylate  were combined with 3 g of ethanol in a closed vial. After stirring at 60 °C for 24 h for the reaction to complete, the solvent was removed via evaporation under vacuum. This process yielded QADM as a clear and viscous liquid. QADM was mixed with the BisGMA-TEGDMA resin at a QADM mass fraction of 20%. A preliminary study has shown that this mass fraction yields strong antibacterial properties without compromising the resin's mechanical properties. This resin is herein referred to as the BisGMA-TEGDMA-QADM resin.The synthesis of antibacterial bis(2-methacryloyloxyethyl) dimethylammonium bromide was as previously described.45 Briefly, 0.1 g of silver 2-ethylhexanoate (0.1 g) was dissolved into 0.9 g of 2-(tert-butylamino) ethyl methacrylate  by gentle stirring. Then, 1% mass fraction of this solution was added to the two resins: the BisGMA-TEGDMA resin and the BisGMA-TEGDMA-QADM resin. This process yielded a 0.1% mass fraction of the Ag salt in each resin. TBAEMA was used because it improved the solubility by forming Ag-N coordination bonds with Ag ions, thereby facilitating Ag salt dissolution in the resin solution. In addition, TBAEMA contains reactive methacrylate groups and can be chemically incorporated into the polymer network upon photo-polymerization.45 The 0.1% mass fraction of silver 2-ethylhexanoate in the resin was used in a preliminary study that showed a strong antibacterial activity, without a noticeable colour change to the resin or negative effects on its mechanical properties.NAg were formed in a dental resin as described previously.n-propylamine.42 Each resin contained a filler mass fraction of 70%, with 20% of NACP and 50% of glass, which were mixed to yield a cohesive composite paste. Because the resin matrix mass fraction was 30%, the QADM mass fraction in the final composite was 6%.To produce NACP composites, three resins were used: (1) the BisGMA-TEGDMA-QADM resin, (2) the BisGMA-TEGDMA-NAg resin, and (3) the BisGMA-TEGDMA-QADM-NAg resin. Each resin was filled with the NACP and with glass particles for reinforcement , which were silanised with 4% 3-methacryloxypropyltrimethoxysilane and 2% Composite containing remineralizing NACP and antibacterial QADM (referred to as NACP+QADM);Composite containing NACP and antibacterial NAg (referred to as NACP+NAg);Composite containing NACP, QADM, and NAg (NACP+QADM+NAg).Therefore, after filling with NACP and glass particles, these three resins resulted in three composites:In addition, a commercial composite served as a comparative control . This resin consisted of fillers of 40 nm to 0.2 micron particle sizes at 60% filler level by volume in a multifunctional acrylic resin of diurethane dimethacrylate and butanediol dimethacrylate. According to the manufacturer, Renamel is indicated for use in Class III, IV, and V restorations.For mechanical testing, each composite paste was placed into rectangular moulds of 2mm × 2mm × 25 mm. For biofilm testing, disk moulds were used with a diameter of 9 mm and a thickness of 2 mm. All specimens were photo-polymerized  for 1 min on each open side of the mould.The specimens were immersed in distilled water at 37 °C for 1 day, 3 months, 6 months, 9 months, or 12 months. This constituted a 4 × 5 full factorial design, with four composites and five water-ageing time periods.−1.42 Flexural strength (S) was calculated as:The water-aged rectangular specimens were fractured using a computer-controlled Universal Testing Machine  in three-point flexure with a span of 10 mm and a crosshead speed of 1 mm·minS=3PmaxL/(2bh2)Pmax represents the load, L represents the span, b represents the specimen width and h represents the thickness. The elastic modulus (E) was calculated as:where E=(P/d)(L3/[4bh3])P divided by displacement d is the slope of the load-displacement curve in the linear elastic region. Six specimens were tested for each composite at each water-ageing time period. The specimens were tested within a few minutes after being taken out of the water, and the specimens were wet while being fractured.where load in vivo plaques.46 Saliva was collected from 10 healthy adult donors possessing natural dentition without active caries or periopathology and who had not used antibiotics within the previous 3 months.48 The donors did not brush their teeth for 24 h and stopped any food/drink intake for at least 2 h before donating saliva. Stimulated saliva was collected during parafilm chewing and kept on ice. An equal volume of saliva from each of the 10 donors was combined to form the saliva sample. The saliva was diluted in sterile glycerol to a saliva concentration of 70%. Aliquots (1 mL) of diluted saliva were stored at −80 °C for subsequent use.48The dental plaque microcosm biofilm model was approved by the University of Maryland Baltimore Institutional Review Board. This model has the advantage of maintaining much of the complexity and heterogeneity of 49 The McBain medium contained mucin  at a concentration of 2.5 g·L−1; bacteriological peptone, 2.0 g·L−1; tryptone, 2.0 g·L−1; yeast extract, 1.0 g·L−1; NaCl, 0.35 g·L−1; KCl, 0.2 g·L−1; CaCl2, 0.2 g·L−1; cysteine hydrochloride, 0.1 g·L−1; haemin, 0.001 g·L−1; and vitamin K1, 0.000 2 g·L−1, at pH 7.49 Each disk was placed into a well of a 24-well plate. Each well was filled with 1.5 mL of the inoculum, and incubated under 5% CO2 at 37 ºC. After 8 h, each disk was transferred to a new 24-well plate with 1.5 mL of fresh medium and incubated under 5% CO2 at 37 °C for 16 h. Then, each disk was transferred to a new 24-well plate with 1.5 mL of fresh medium and incubated for 24 h. This totalled 2 days of incubation, which has been shown to be sufficient to form relatively mature biofilms on dental resins.33Composite disks after water-ageing were sterilized with an ethylene oxide sterilizer  and then degassed, following the manufacturer's instructions. The saliva-glycerol stock was added, using 1:50 final dilution, to a McBain artificial saliva growth medium as the inoculum.48 Imaging was performed via a confocal laser scanning microscope . Four randomly chosen fields of view were photographed for each disk, with six disks per material at each time period, yielding 24 images for each composite at each water-ageing time period.The composite disks with 2-day biofilms were rinsed with phosphate-buffered saline (PBS) and live/dead stained using the BacLight live/dead bacterial viability kit .28 MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to purple formazan. Disks with 2-day biofilms (n=6) were transferred to a new 24-well plate, and 1 mL of MTT dye (0.5 mg·mL−1 MTT in PBS) was added to each well and incubated at 37 °C under 5% CO2 for 1 h. During the incubation, metabolically active bacteria metabolized the MTT to formazan inside the living cells. Disks were then transferred to new 24-well plates, and 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. The plates were incubated for 20 min with gentle mixing at room temperature. Two hundred microlitres of the DMSO solution from each well was collected, and the absorbance at 540 nm was measured via a microplate reader . A higher absorbance indicates a higher formazan concentration, which is related to more metabolic activity in the biofilm on the composite surface.33A 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the metabolic activity of the biofilms.n=6) were rinsed with cysteine peptone water (CPW) to remove loose bacteria, and then transferred to 24-well plates containing 1.5 mL of buffered-peptone water (BPW) plus 0.2% sucrose. The specimens were incubated for 3 h to allow the biofilms to produce acid. Then, the BPW solutions were collected for lactate analysis using an enzymatic method.33 The 340-nm absorbance of BPW was measured with a microplate reader . Standard curves were prepared using a standard lactic acid  as described in previous studies.33Disks with 2-day biofilms , by following the methods used in previous studies.33 Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine the total number of microorganisms.33 Second, mitis salivarius agar (MSA) culture plates, containing 15% sucrose, were used to determine the total number of streptococci.50 This count was feasible because MSA contains the selective agents crystal violet, potassium tellurite, and trypan blue, which inhibit most Gram-negative bacilli and most Gram-positive bacteria except streptococci, thus enabling streptococci to grow.50 Third, cariogenic mutans streptococci are known to be resistant to bacitracin, and this property is often used to isolate mutans streptococci from the highly heterogeneous oral microflora. Hence, MSA agar culture plates plus 0.2 units of bacitracin per mL were used to determine the number of mutant streptococci.51 The bacterial suspensions were serially diluted and spread onto agar plates for colony-forming unit (CFU) analysis by following the methods used in previous studies.Disks with 2-day biofilms were transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication and vortexing P-value of 0.05.Statistical analyses were performed using Statistical Package for the Social Sciences . Two-way analyses-of-variance (ANOVA) were performed to detect the significant effects of the variables. Tukey's multiple comparison was used to compare the data at a n=6). For all four composites, there was a moderate decrease in strength and modulus from 1 day to 3 months of water-ageing (P<0.05). There was little subsequent decrease from 3 to 12 months (P>0.1). At each water-ageing time period, the flexural strength and elastic modulus of NACP-QADM, NACP-NAg, and NACP-QADM-NAg composites were similar to those of the commercial control composite (P>0.1).The flexural strength and elastic modulus results are plotted in Representative live/dead images of 2-day biofilms on the composites are shown in P<0.05). Furthermore, incorporating QADM and NAg together into the same composite resulted in the lowest metabolic activity and lactic acid production (P<0.05). For all composites, the biofilm metabolic activity was similar from 1 day to 12 months of water-ageing (P>0.1), and the lactic acid production from 1 day to 12 months was also not significantly changed over this period (P<0.05). These results demonstrate that incorporating QADM and NAg into the NACP composite substantially decreased the biofilm metabolic activity and lactic acid production, and the antibacterial potency was maintained and durable during water-ageing for 1 year.n=6). Adding QADM or NAg significantly reduced the CFUs compared with the commercial composite control, and combining both QADM and NAg into the same composite yielded the least CFU counts. For each composite, the CFU counts of biofilms did not vary significantly vs water-ageing time from 1 day to 12 months. For example, the NACP-QADM-NAg composite had a similar number of CFUs at 1 day and after 3, 6, 9, or 12 months of water-ageing (P>0.1).The CFU counts for 2-day biofilms on composite disks are plotted in 38In the present study, antibacterial and remineralizing dental composites were developed containing QADM, NAg, and NACP, which were demonstrated to maintain the anti-biofilm activity after 12 months of water-ageing. Incorporating QADM and/or NAg into the NACP composite did not decrease its mechanical properties, which matched those of a commercial composite control after water-ageing for 1 year. Quaternary ammonium salts are important compounds with a broad antimicrobial spectrum. The advantage of QAM-containing dental resins is that the antibacterial monomer is copolymerized with the resin matrix by forming a covalent bond with the polymer network, and therefore, the QAM is immobilized in the resin and is not released or lost over time. Therefore, this copolymerization and immobilization method may impart a durable antibacterial capability to the dental resin. The present study provides the first demonstration that after 1 year of water-ageing, the antibacterial potency of a composite containing QADM, NAg, and NACP is still as strong as it was at 1 day. Such sustained reduction in biofilm/plaque and acid production may help minimize tooth structure demineralization, and the addition of NACP may promote remineralization.28 The synthesis of QADM involves a modified Menschutkin reaction in which a tertiary amine group is reacted with an organo-halide, and the reaction products are generated at virtually quantitative amounts and require minimal purification.28 In addition, as a dimethacrylate, QADM can be beneficial as compared as quaternary ammonium monomethacrylates. QADM has reactive groups on both ends of the molecule, which can be incorporated into the resin matrix with less of a negative effect on the mechanical and physical properties of the composite.28 In contrast, a quaternary ammonium monomethacrylate has only one reactive group and may weaken the resin matrix when incorporated. Furthermore, QADM is compatible with common dental dimethacrylate monomers and is expected to have minimal monomer leachability, compared with other quaternary ammonium salts. The detailed antimicrobial mechanism of quaternary ammonium remains to be fully understood; however, quaternary ammonium materials appear to cause bacterial lysis by binding to the cell membrane and causing cytoplasmic leakage.25 This leakage is stimulated when the negatively charged bacterial cell contacts the positively charged (N+) sites of the quaternary ammonium resin. This disturbs the electrical balance of the cell membrane, and the bacterium then ruptures under its own osmotic pressure. The results of the present study involving water-ageing for 1 year showed that such an antibacterial mechanism indeed has a long term and durable inhibitory effect on oral biofilm growth.The present study used QADM because its synthesis method is fairly straightforward as compared with the synthesis of other QAMs.52 Its antimicrobial mechanism appears to involve Ag ions interacting with and inactivating the vital enzymes of bacteria, causing the DNA in the bacteria to lose its replication ability, leading to cell death.52 Although the present study indicates that the NAg in the composite possesses antibacterial activity even after 12 months of water-ageing, further studies are needed to investigate how long the antibacterial effect of NAg resin lasts and whether the antibacterial potency decreases after 2 or 3 years. Furthermore, the results of the present study showed that the use of two antibacterial agents (QADM+NAg) achieved a significantly stronger antibacterial potency than using either single agent, and the activity of the dual agents was maintained after water-ageing for 1 year. This was achieved because both QADM and silver, through TBAEMA's reactive methacrylate groups, which copolymerize with the resin matrix.45 These results suggest that copolymerization with the resin matrix and the use of dual agents appear to be promising approaches to maximize the antibacterial function of the resin without compromising its mechanical properties or long-term durability.Ag is another effective and widely used antibacterial agent with a strong toxicity to a wide range of microorganisms.53 In contrast, commercial restorative controls fail to raise the pH.53 Other studies have shown that the NACP composite remineralizes enamel lesions38 and inhibits secondary caries at the composite-enamel margins in the oral environment, in a human in situ model.54 The NACP composite in these previous studies had no antibacterial agent in the composite. In the present study, incorporating both QADM and NAg into the NACP composite achieved double benefits: remineralization capability due to NACP and anti-biofilm activity due to QADM and NAg. Therefore, we expect that its caries-inhibition efficacy will be further enhanced when compared with NACP alone. Further studies are needed to compare the inhibition of secondary caries at the tooth-restoration margins by NACP vs NACP+QADM+NAg in a human in situ model.In addition to the QADM and NAg incorporation reducing biofilm acids and combating caries, NACP incorporation may also contribute to the inhibition of caries. A previous study has shown that a NACP composite can release Ca and P ions and neutralize an acid, rapidly increasing the pH of an acidic solution from a pH of 4 to one above 6.55 Gingival recession exposes more tooth roots, which have a higher solubility and are less resistant to biofilm acids than tooth enamel.56 In addition, reduced saliva flow tends to increase biofilm and plaque buildup.57 Indeed, root caries increase from 7% among young people to 56% in seniors.58 Class V restorations with subgingival restorative margins have pockets for bacterial growth that are difficult to clean and can lead to periodontitis.59 Therefore, there is a need to develop novel bioactive restoratives for Class V lesions that are antibacterial to inhibit cariogenic biofilms as well as periodontal pathogens in cases with subgingival margins. The antibacterial function of the NACP+QADM+NAg composite, as well as the ability of NACP to neutralize acids and to raise the pH above 6  in the same composite resulted in a significantly stronger antibacterial capability than using QADM or NAg alone. The antibacterial results were not significantly different after water-ageing for 12 months, compared with 1 day, thus indicating a long-term durability. The durable antibacterial and mechanical properties, plus the calcium and phosphate ion release and acid neutralization reported previously for remineralization, indicate that the novel NACP-QADM-NAg composites may be useful in dental restorations to combat caries.


Direct growth of spherical graphene with large surface area is important for various applications in sensor technology. However, the preparation of transfer-free graphene on different substrates is still a challenge. This study presents a novel approach for the transfer-free graphene growth directly on cemented carbide. The used simple thermal annealing induces an in-situ transformation of magnetron-sputtered amorphous silicon carbide films into the graphene matrix. The study reveals the role of Co, a binding phase in cemented carbides, in Si sublimation process, and its interplay with the annealing temperature in development of the graphene matrix. A detailed physico-chemical characterisation was performed by structural , morphological (SEM) and chemical (EDS) analyses. The optimal bilayer graphene matrix with hollow graphene spheres on top readily grows at 1000 °C. Higher annealing temperature critically decreases the amount of Si, which yields an increased number of the graphene layers and formation of multi-layer graphene (MLG). The proposed action mechanism involves silicidation of Co during thermal treatment, which influences the existing chemical form of Co, and thus, the graphene formation and variations in a number of the formed graphene layers. The transfer of graphene generates impurities and defects that vitiate the mechanical and electrical properties3. Another problem is low bonding strength between graphene and different substrates.Synthesis of graphene on different substrates is a challenging and attractive approach that can broaden application spectra of currently existing engineering materials. Versatile experimental approaches were used, but major constraints are related to synthesis of adequate graphene sheets and subsequent application problems caused by the transfer of synthesised grapheneet al.4 reported graphene formation on Ni surface by the mechanism of carbon dissolving from SiC substrates by rapid heating. Machač et al.5 demonstrated a feasible route for graphene growth by annealing of Ni/SiC structures, highlighting the role of Ni layer thickness and a shortage of Ni amount in the structure. Surface Ni-silicidation reaction is also an effective mean for preparation of few-layer graphene (FLG) by controlled thermal annealing of SiC crystals, which was successfully demonstrated in Ni/6H-SiC (0001) system, with the best graphene structure obtained at 800 °C6. The similar approaches are also valuable for the transfer-free synthesis of graphene on SiO27, sapphire and Cu2O (111) substrates8. Besides Ni and Cu, another transition metal, Co, was investigated as an attractive catalyst for transfer-free graphene growth. By deposition of Co layer on SiC, the FLG was grown via selective reaction between Co film and SiC substrate at high temperatures followed by the rapid cooling9. The question that arises here is related to control of carbon diffusion process since it affects number and quality of graphene layers; particularly, formation of the top CoO layer is actually critical for the synthesis of a high-quality graphene layer10. Having in mind these results, a successful synthesis of transfer-free graphene layer by the metal-silicidation approach requires several steps, including deposition of selected metal layer/metal oxide layer, control of processing parameters and carbon diffusion process as well.Metal-catalysed graphene growth from SiC followed by a simple but controlled annealing step is a useful approach towards the synthesis of large area graphene layer. Among different metals, Ni, Cu and Co effectively catalysed graphene generation. Juang 12. Development of a suitable material on the surface of cemented carbide tools could enable acquisition of drilling information, detection and automated correction of certain failure modes, such as auger choking, auger jamming, and bit wear15. Among different materials, graphene-based composites due to their remarkable mechanical, electrical, piezo-resistive and other physical properties become the best option regarding the inherent conventional sensor defects, such as low sensitivity, short service life, and rapid attenuation19. Especially three-dimensional (3D) graphene with hollow spheres on the surface shows enhanced sensing performances due to large surface area that provides more binding sites, which has great application potential20. However, there are no acceptable methods to synthesise in-situ a graphene layer on the cemented carbide surface, especially in regards to following aspects: (1) using readily available a-SiC instead of costly SiC single-crystal as carbon source, (2) the impact of annealing temperature on the number of produced graphene layers and (3) the understanding of Si atoms role in graphene growth and change in the number of graphene layers. If the mechanism of Si acting is not identified, the graphene layercannot be directly grown from a-SiC.Cemented carbides are extensively used materials in space drilling applications for providing a first-hand insight into the geological structure and mineral distribution of planets but also as cutting tool materials22. The clear advantage of this study is an elegant approach towards utilisation of currently existing engineering material and scientific data to develop a sustainable new aspect of cemented carbides without any need of depositing metal oxide layer to control carbon diffusion. The graphene layer with graphene balls on top is formed in-situ by simple and controlled annealing. An extensive physicochemical characterization, including structural, morphological and chemical aspects, was performed to reveal the influence of temperature on the number of formed graphene layers and mechanism of Si atom acting during the graphene matrix generation. This paper opens new horizons towards further enhanced industrial applications of the cemented carbides.In this study, we propose a novel approach towards the in-situ synthesis of graphene layer on cemented carbides by utilisation of Co as a binding phase in cemented carbides to catalyse the in-situ growth of the graphene film. Herein, Co binding phase catalyses graphitization of amorphous SiC (a-SiC), which is deposited on the cemented carbide at low temperature by the magnetron sputtering technique. In addition, this does not change the carbide performance due to a trace amount of Co involvedGraphene sheets with graphene balls on top were grown directly on the cemented carbide surface by a new approach that uses Co as a binding phase in the cemented carbides to catalyse the formation of graphene from a-SiC. Moreover, the exact mechanism of graphene formation by annealing a-SiC was revealed. The influence of annealing temperature on morphology, film composition, structural changes and number of graphene layers was systematically studied. Furthermore, the role of Si atoms in graphene formation was discussed regarding the phase transformations and the number of graphene layers.23.In the present study, Co from cemented carbides is used to in-situ catalyse graphene formation from magnetron-sputtered a-SiC films by an annealing process. The Raman spectra of the deposited a-SiC films annealed at five different temperatures are shown in Fig. 2D/IG), full width at half maximum (FWHM) of the peak and the number of fitted Lorentzian peaks24. The general equation for determination of the crystallite size, La using any laser line in the visible range can be written as25:λ is 532 nm. The Raman spectrum at annealing temperature of 1000 °C showed that I2D/IG value was higher than 1, FWHM was 45 cm−1 and the 2D peak was at 2700 cm−1. For a single layer, the intensity of the 2D peak was four times higher than that of the G peak, whilst the intensity of the 2D peak was 1 to 2 times higher than that of the G peak for two layers26; FWHM of the 2D peak for a single layer of graphene is in the range of 30–45 cm−1, for two layers is in the range of 45–60 cm−1 and for three layers is in the range of 65–70 cm−1, whilst the FWHM of the 2D peak of graphite was lower than 30 cm−1 27. Inset 2-a indicates that 2D peak may be deconvoluted into four peaks with maxima at 2659, 2687, 2704 and 2725 cm−1, respectively. In general, a 2D peak of a single layer of graphene can be fitted by a single Lorentzian profile, a 2D peak of two layers of graphene can be fitted by four Lorentzian profiles, and a 2D peak of three layers of graphene can be fitted into six Lorentzian profiles, whilst a 2D peak of four or more graphene layers can be fitted only with two Lorentzian profiles23. Therefore, it can be concluded that by annealing at 1000 °C, the quality graphene with 2 layers was successfully generated on the cemented carbide.Data of the D, G and 2D peaks of the graphene obtained at 1000 and 1150 °C are listed in Table −1 (Inset 2-b), respectively. It is suggested that the number of graphene layers was increased. In addition, the crystallite size of graphene (La) decreased and the ID/IG ratio increased, indicating that the number of film defects increased with the annealing temperature28. The reason for the increase of a number of defects with temperature is the temperature increase leads to the increase in the number of graphene layers. This induces the formation of boundaries among the graphene layers and the increase of the ratio of ID/IG.The intensity ratio of the 2D and G peaks at annealing temperature of 1150 °C is lower than that at 1000 °C, whilst the FWHM of the former is greater than of the latter; thus, it is clear that graphene is generated, but the number of layers produced at 1150 °C is obviously higher. The 2D peak may be fitted by two Lorentzian profiles with maxima at 2689 and 2710 cm2D/IG ratio and FWHM of Raman mapping of the graphene film synthesised at two annealing temperatures, 1000 and 1150 °C, with the size of 100 × 100 μm2. As shown in Fig. 2D/IG ratio is in the range of 1.1–1.5, and FWHM, Fig. −1. At 1150 °C, Fig. 2D/IG ratio value drops and ranges from 0.7 to 1.2, whilst FWHM value, Fig. −1 but also less than 30 cm−1. From these findings can be seen that the graphene film has high quality and consists of 2 layers at 1000 °C, whilst at 1150 °C, the film consists of several layers of graphene and graphite phases.Figure Thus, in the suitable annealing temperature range (1000–1150 °C), the graphene film was formed on the cemented carbide by Co-catalysed a-SiC transformation. When the annealing temperature was increased from 1000 to 1150 °C, the number of graphene layers, as well as the number defects of the graphene films, were increased, and the quality of the graphene layer was degraded.The number of graphene layers is an important parameter to distinguish between graphene and graphite phases and to gauge the quality of graphene. Therefore, it was attempted to investigate how the annealing temperature influences the number of graphene layers. The following approaches were applied: (1) probing the impact of the annealing temperature on the graphene layer structure by XRD; (2) SEM monitoring of morphological changes of the graphene layer upon the annealing process; and (3) analysis of the annealing temperature impact on the graphene film composition by EDS.2Si and Co, respectively, appeared in the XRD spectra, denoting that within this temperature range Co diffused from cemented carbide into the SiC film and reacted with SiC, thus preventing the crystallisation of a-SiC. However, the diffraction peak of β-SiC appeared on the surface of the Si slice at 1000 and 1150 °C,  is a thermodynamically spontaneous reaction. If the reaction temperature is higher than 600 °C, Co2Si is generated (2Co + SiC → Co2Si + C), and if the temperature is higher than 800 °C, the materials further react to form CoSi (Co2Si + SiC → 2CoSi + C)29. The formation of graphene is based on the reaction mechanism where C is isolated on the surface of the Co/CoxSiy film, owing to the decrease in solubility of C with temperature decrease30. As for the annealing temperature of 1000 °C, the Raman analysis shows that graphene was synthesised from the a-SiC film, which is the process catalysed by Co. Because the number of graphene layers was low, the graphite phase was not detected by XRD at approximately 26.5 ° (2θ). The Co reacts with the a-SiC film to form Co2Si, and the Co/Si atomic ratio after reaction may contribute to this reaction product31. When the Co/Si atom ratio increases, a compound phase in the Co-Si bi-component system approaches Co, and the CoSi compound transforms to Co2Si28. Thus, at the annealing temperature of 1000 °C, Co sufficiently reacts with the a-SiC film to catalyse graphene generation, generating carbon-saturated Co2Si.Different products were generated at different annealing temperatures by the catalytic reaction of Co and a-SiC. The structure of precipitated graphene layer was also changed with temperature. Systematic research on the impact of the annealing temperature on the structure of precipitated graphene layer could assist in revealing the mechanism by which the temperature causes the change in the number of graphene layers. Upon heating, Co and SiC interact, generating the Co-Si compound saturated with C atoms2Si compound. The same as afore-mentioned, Co reacts with the a-SiC film at 800 °C to preferentially generate CoSi. At the annealing temperature of 1150 °C, the CoSi compound in the film disappeared, and the Co phase appeared. Meanwhile, the number of the generated graphene layers increased and the graphite phase could be also detected.Unlike that at 1000 °C, Co reacts with a-SiC at 1150 °C, and there is only the Co phase without Co2Si compound saturated with C atoms, and the quality graphene layer with low ID/IG (0.339) and high I2D/IG (1.304) ratio is obtained. When the annealing temperature is further increased, the Co2Si compound transforms into Co, and the amount of precipitated C is increased, as well as the number of graphene layers.Therefore, in the suitable annealing temperature range (1000–1150 °C), Co diffuses in a-SiC film, catalysing graphene formation. At 1000 °C, Co reacts with a-SiC to form the CoThe impact of the annealing temperature on graphene morphology is shown in Fig. 32.The presented results show that at annealing temperatures of 1000 and 1150 °C, Co diffused into a-SiC film and catalysed the formation of graphene thereon. The metal reacted with a-SiC film and metal silicide spontaneously agglomerated to reduce the surface energy, which is in agreement with previously reported results that the graphene film is generated preferentially at areas where the metal disappeared after contraction2Si particles agglomerated on cemented carbide; hollow graphene balls were generated and graphene sheets with graphene balls were obtained. When the annealing temperature was increased to 1150 °C, Co2Si particles disappeared, the film of hollow graphene balls became thicker and the number of graphene layers increased. Such graphene sheets, with hollow graphene balls that meet the requirement of the high specific surface area for an ideal graphene film, can be beneficial for improvement of the efficiency of strain sensors by the reduction of size and power consumption23.Thus, it can be concluded that at the annealing temperature of 1000 °C, the graphene balls were precipitated from Co2Si compound disappeared, so the Co phase appeared in the film, and the number of generated graphene layers increased. In the Co-Si system, different Co/Si ratios resulted in different Co-Si phases29. The mechanism by which the precipitation layer structure of segregated C was altered with temperature may be revealed only by a systematic research of the impact of the annealing temperature on the atomic content during the synthesis of graphene. Figure 2Si and the reaction with SiC is not possible, so it does not appear in the a-SiC film. When the annealing temperature was increased, Co atoms appear in the film, the content of Si atoms decreases and the content of C atoms becomes higher, which implies that Co diffused into the a-SiC film.Influence of annealing temperature on the chemical composition of graphene film was monitored by EDS. In the temperature range of 1000–1150 °C, when the annealing temperature was increased, the Co–4 Pa) conditions for Si sublimation from SiC film to enable restructuring of C atoms and graphene formation, graphene can be synthesised at low temperature using a SiC film with Co catalyst33. This study shows that at 1000 °C, Co diffused into the a-SiC film and at Co concentration of 11.43%, the content of Si dropped from 30.5% to 4.22%, whilst the amount C atoms increased to 84.35%. It suggests that Co catalysed the sublimation of Si and the restructuring of C atoms, decreasing the content of Si and rearranging C atoms to generate graphene. Compared to that, at 1150 °C the content of Co atoms remained essentially unchanged, 10.83%, the amount of Si was even lower, 0.93% and the amount of C atoms increased to 88.24%. Having in mind the afore-mentioned results of Raman and XRD measurements, it can be stated that at the annealing temperature of 1150 °C, the Co-catalysed sublimation of Si was accelerated, inducing Co2Si cracking and the increase in a number of segregated carbon atoms in Co layer, as well as the number of graphene layers.Unlike conventional graphene epitaxial growth by SiC that requires high temperatures (>1200 °C) and high vacuum  graphene is directly generated from the a-SiC film on the surface of cemented carbide catalysed by Co binding phase via the annealing process in the temperature range of 1000–1150 °C. When the annealing temperature is increased (1150 °C), the number of graphene layers increases and the graphene quality becomes poorer. (2) Influence of the annealing temperature on the number of graphene layers is related to changes in the film composition and structure. In the suitable temperature range (1000–1150 °C), Co catalyses Si sublimation, and C particles segregate to generate graphene. The related changes in atomic and phase compositions at the higher annealing temperature induced the increase in the number of graphene layers. (3) The content of Si influences the Co pattern and causes the generation of graphene and change in the number of graphene layers. The mechanism of Si atoms acting actually includes three subsequent parts: (a) when the content of Si particles decreases, C particles segregate in the Co layer to generate graphene; (b) Si particles react with Co to generate Si-Co compound, so the free Co layer disappears, and the number of precipitated graphene layers is low; and, (c) when Si particles disappear, no reaction with free Co metal occurs, and the number of graphene layers increases.This research study provides a new deep insight into the transfer-free direct growth of graphene on the surface of cemented carbides, explaining the mechanism of chemical processes behind, especially underlying the structural, chemical and morphological aspects of the graphene layer formation.−3 Pa), Ar ion bombardment duration (20 min), bias (−800 V), deposition pressure (0.5 Pa), C2H2 flow rate (35 SCCM), substrate bias (−100 V), target current (22 A) and deposition temperature (150 °C). The film thickness was approximately 2.0 μm.The a-SiC films were deposited by a mid-frequency magnetron sputtering technique. There were two kinds of substrate materials: monocrystal Si wafer was used for control analysis of the structure, whilst cemented carbide YG 8 (WC-Co 8%) was used for the analysis of the graphene structure. The sample preparation method included following steps: (1) the substrate was cleaned in both acetone and absolute ethanol by ultrasonication in each medium for 10 min, and (2) the substrate was dried by nitrogen and placed in a vacuum chamber for deposition. The reaction of Si target and acetylene, which provided the source of silicon and carbon atoms, respectively, was used for the a-SiC film deposition. The main experimental parameters include basic pressure , 2θ range 20–80° and a step size of 0.02°). Valence bond and the number of layers of graphene films were determined by Raman spectroscopy  using 532 nm laser excitation at room temperature, and layer uniformity is revealed by Raman mapping; surface morphology was identified by scanning electron microscopy , while compositions of the film surfaces were analysed by energy-dispersive X-ray spectrometry (EDS). The atomic composition was obtained by taking the average of six measurements on the surface of the individual sample.


Using the ultrafast pump-probe transient absorption spectroscopy, the femtosecond-resolved plasmon-exciton interaction of graphene-Ag nanowire hybrids is experimentally investigated, in the VIS-NIR region. The plasmonic lifetime of Ag nanowire is about 150 ± 7 femtosecond (fs). For a single layer of graphene, the fast dynamic process at 275 ± 77 fs is due to the excitation of graphene excitons, and the slow process at 1.4 ± 0.3 picosecond (ps) is due to the plasmonic hot electron interaction with phonons of graphene. For the graphene-Ag nanowire hybrids, the time scale of the plasmon-induced hot electron transferring to graphene is 534 ± 108 fs, and the metal plasmon enhanced graphene plasmon is about 3.2 ± 0.8 ps in the VIS region. The graphene-Ag nanowire hybrids can be used for plasmon-driven chemical reactions. This graphene-mediated surface-enhanced Raman scattering substrate significantly increases the probability and efficiency of surface catalytic reactions co-driven by graphene-Ag nanowire hybridization, in comparison with reactions individually driven by monolayer graphene or single Ag nanowire. This implies that the graphene-Ag nanowire hybrids can not only lead to a significant accumulation of high-density hot electrons, but also significantly increase the plasmon-to-electron conversion efficiency, due to strong plasmon-exciton coupling. The hot carriers then redistribute their energy through electron–electron scattering processes on a timescale τel ranging from 100 fs to 1 ps352 hybrid had been used to study the plasmon-driven chemical reaction10122 nanoparticles, by using femtosecond transient absorption spectroscopy with an IR probe2 nanoparticles, and the reaction time was within 240 fsSurface plasmon resonance (SPR) is the resonant collective oscillation of conduction electrons at the interface between materials with negative and positive permittivities, which is stimulated by incident light3Graphene, a single atomic layer of graphite, has attracted vast interest owing to its unique properties since its discovery in 200415171819202122232526In this paper, novel optical graphene-Ag nanowire hybrid materials are fabricated, and the advantages of this hybrid device are shown through ultrafast pump-probe transient absorption spectroscopy and surface catalytic reactions on this hybrid nanostructure. The experimental results indicate that graphene can strongly harvest hot electrons generated from Ag plasmon decay, which can not only lead to a significant accumulation of high density hot electrons, but also prolong the lifetime of these hot electrons from femtosecond to picosecond, through Ag plasmon-enhanced graphene plasmon. Furthermore, the surface-plasmon-to-hot-electron conversion efficiency is significantly enhanced. Our results show that the graphene-Ag nanowire hybrids with plasmon-exciton interaction can act as a good candidate in spectral censoring, catalysis, and potential applications in nano-photonic devices, energy and environments.2/Si substrate is shown in 2D/IG ≈ 2.23)The SEM image of the graphene on SiO2 nanoparticles in IR regionThe SEM image of a single Ag nanowire coated by monolayer graphene can be seen in F < ε < EF + hv in the system, where EF is the Fermi energy level.We also measured the time of charge transfer between Ag nanowire and single layer graphene in the NIR region, as shown in 32There are two dynamic processes with different time scales in the NIR region. The fitted time of hot electrons transferring from Ag nanowire to graphene is about 780 ± 92 fs. It is found that the reaction time of hot electron from Ag nanowire to graphene in the NIR region is slower that in the VIS region, due to lower kinetic energy in the NIR region. The fitted curve in One of the advantages of graphene-Ag nanowire hybrids is that, graphene plasmon enhanced by metal plasmon can be used to drive chemical reactions on this hybrid nanostructure The surface catalytic reaction of 4-nitrobenzenethiol (4NBT) to p,p’-dimercaptoazobenzene (DMAB) without graphene has already been clearly investigated−1 to 1500 cm−1 in To highlight the advantages of plasmon-graphene hybrids for the catalytic reactions, we first measured the Raman spectra of 4NBT and DMAB powders, and SERS spectra of 4NBT at the junction of tip-to-tip Ag nanowires  cannot be observed because of the strong background of graphene. We propose that photo-induced hot electrons generated from graphene provide the kinetic energy and the electrons for the reduction reactions from 4NBTs to DMAB. The density and efficiency of hot electrons provided by the graphene are strongly dependent on the laser power. However, the intensity of Raman spectra in the chemical reactions is weak, compared with the strong graphene Raman signal. More electrons with higher kinetic energy are needed to increase the reaction probability and efficiency of graphene-driven reduction reactions when excited with weak laser power.Now we present the results of our investigation on the graphene-driven reduction reactions of 4NBTs to DMAB on the GERS substrate. The SEM image is shown in −1 corresponding to the N-O2 vibrational modes of 4NBT almost disappears completely), which exhibits that the plasmon-graphene hybrids can provide a high-density hot electrons, and thus that leads to a highly efficient catalytic reaction. It is clearly observed that the intensities of Raman peaks of graphene at 1595 and 2687 cm−1 gradually increase with the increase of the laser power. When the laser power reaches 100% (11 mW), the Raman intensity at 2687 cm−1 becomes larger than that at 1595 cm−1, I2D/IG ≈ 1.13. Thus, we conclude that 1) the catalytic reaction is already highly efficient at weak laser power, and the reaction probability can reach 100% by increasing the laser power; 2) the intensities of the graphene Raman peaks at 1595 and 2687 cm−1 also increase gradually, and for sufficiently high laser intensities, the Raman intensity at 2687 cm−1 become larger than that at 1595 cm−1, which demonstrates that the catalytic reaction indeed occurs on the monolayer graphene-coated Ag nanowires. Note that Raman intensities of DMAB are weaker compared with the Raman intensities of graphene, the reason is that some of the DMAB may be desorbed from the surface of graphene when radiated at enough strong laser. There are two superposed Raman peaks between graphene and DMAB around 1595 cm−1, which leads to I2D/IG > 1, but is less than 2.3, when the laser power is 100%.The SEM image of graphene-Ag nanowire hybrids is presented in Novel graphene-Ag nanowire hybrid optical materials are fabricated and probed using the ultrafast pump-probe transient absorption spectroscopy, and then exploited as the G-SERS substrates to investigate the typical surface reductions of 4NBTs to DMAB. Firstly, single-layer graphene can significantly improve the surface-plasmon-to-hot-electron conversion efficiency. Secondly, with the assistance of graphene, the lifetime of hot electrons is greatly increased from femtosecond to picosecond. As a consequence, this G-SERS substrate significantly increases the probability and efficiency of the surface catalytic reactions of 4NBTs to DMAB co-driven by graphene-Ag nanowire hybridization, compared with the same kinds of reactions but driven individually either by graphene or single Ag nanowires. The graphene-Ag nanowire hybrids, with higher conversion efficiency and longer lifetime of plasmon-induced hot electrons, can be used as a source of hot electrons for catalysis and might be potentially applied as an analytical tool for observing such plasmon-driven reactions.2 gas at 1000 °C for 5 min. In the initial stage of the reaction, the reaction gaseous mixture Ar:H2:CH4 with a flow rate of 960: 40: 20 sccm was introduced into the tube. The reaction took place for 5 min at 1000 °C. Finally, the CVD tube was cooled to 150 °C under an Ar:H2 environment.Large-area monolayer graphene was synthesized on copper foils  using cold-wall low-pressure chemical vapour deposition (CVD)2/Si substrate and then dried in the air at room temperature.The silver nanowires were prepared through chemical fabrication2/Si substrate. First, a PMMA layer was spin-coated  on the graphene-covered copper foil. The copper foil was then etched away by ferric chloride solution (0.5 M) for 4 h. The PMMA film with graphene was soaked in deionized water four times and then transferred from the water to the substrate with Ag nanowires. Finally, the PMMA was dissolved in an acetone solution and then the sample was washed in an ethanol and deionized water.Polymethyl methacrylate (PMMA) was used to help transfer the graphene from the copper foil to Ag nanowires on the SiO−5 M 4-NBT ethanol solution for more than 12 h. The substrates were then washed with ethanol for 5 min, and dried with N2 gas. All SERS spectra were measured by using a Renishaw inVia confocal Raman spectrometer with a 532 nm laser  as the excitation light. Each Raman spectrum was recorded with an accumulation time of 10 s.The G-SERS substrates were immersed in a 5 × 10ex = 400 nm) of the fundamental laser by a 0.5 mm BBO . The 400 nm beam was attenuated to a power of approximately 3 μJ per pump pulse at the sample. The excited states were probed by using the other beam generated to pass through a controlled delay line  and focus onto a sapphire plate to generate a sub-picosecond white-light super-continuum. The pump and probe beams were incident on the sample at a small angle (θ ≤ 5°). Samples were placed in the beam path where the beam diameter was 300 μm. All of the above experimental measurements were performed at room temperature (295 K). The sample of Ag nanowire-graphene was placed on the quartz substrate for the transient absorption spectroscopy.The morphologies of the substrate system consisting a single-layer graphene-Ag nanowire were characterized by using a scanning electron microscope . The setup of the transient absorption spectroscopy has been reported in the previous paperHow to cite this article: Ding, Q. et al. Ultrafast Dynamics of Plasmon-Exciton Interaction of Ag nanowire-Graphene Hybrids for Surface Catalytic Reactions. Sci. Rep.6, 32724; doi: 10.1038/srep32724 (2016).


The APOBEC3 family of cytosine deaminase enzymes are able to restrict replication of retroelements, such as LINE-1. However, each of the seven APOBEC3 enzymes have been reported to act differentially to prevent LINE-1 retrotransposition and the mechanisms of APOBEC3-mediated LINE-1 inhibition has not been well understood. The prevailing view for many years was that APOBEC3-mediated LINE-1 inhibition was deamination-independent and relied on APOBEC3s blocking the LINE-1 reverse transcriptase DNA polymerization or transport of the LINE-1 RNA into the nucleus. However, recently it was shown that APOBEC3A can deaminate cytosine, to form uracil, on transiently exposed single-stranded LINE-1 cDNA and this leads to LINE-1 cDNA degradation. In this study, we confirmed that APOBEC3A is a potent deamination-dependent inhibitor of LINE-1 retrotransposition, but show that in contrast, A3H haplotype II and haplotype V restrict LINE-1 activity using a deamination-independent mechanism. Our study supports the model that different APOBEC3 proteins have evolved to inhibit LINE-1 retrotransposition through distinct mechanisms. Transposable elements can be grouped into two major classes based on whether they mobilize via a DNA intermediate  or an RNA intermediate 2. DNA transposons, although not being able to mobilize in the current human genome, have played a major role during the evolution of the eukaryotic genome6. Retrotransposons transpose through mRNA intermediates followed by reverse transcription, synthesis of double-stranded (ds) DNA, and integration at new genomic locations. Retrotransposons can be divided into two classes: Long Terminal Repeat (LTR) retrotransposons  and the non-LTR retrotransposons. The replication strategy of endogenous retroviruses resembles those of retroviruses transmitted person to person, except that they remain in one host and are transmitted vertically2. The present day endogenous retroviruses have acquired so many mutations that they cannot produce infectious virus2.Transposable elements are DNA sequences that can move from one location of the genome to another. Approximately 45% of the human genome is recognized as being derived from transposable elements8. Although the vast majority of L1s are inactive due to accumulated mutations or 5′ truncations, the human genome still contains ~80–100 copies of intact L1s that are retrotransposition competent10. Full-length L1 contains a 5′ UTR, two Open Reading Frames (ORFs) that are separated by a 63 nt inter-ORF region, and a 3′ UTR that ends with a polyadenosine-rich sequence (poly(A) tail)11. Proteins encoded by ORF1 and ORF2 are both required for efficient L1 retrotransposition. ORF1 encodes a nucleic acid binding protein with demonstrated nucleic acid chaperone activity; ORF2 encodes proteins with endonuclease (EN) activities and reverse transcriptase (RT) activities14. L1-encoded EN preferentially cleaves ssDNA at an AT-rich consensus sequence . L1 RT is a DNA/RNA dependent DNA polymerase, but lacks RNaseH activity, a trait that distinguishes itself from the retroviral RTs15. As a result, during cDNA synthesis the RNA strand of the resulting L1 RNA/DNA hybrid could be either degraded by a yet unidentified cellular protein or displaced during (+) strand cDNA synthesis16. Cellular RNaseH2 cooperates with flap endonuclease 1 (FEN-1) to remove RNA primers during lagging strand DNA synthesis and serves as a good candidate17. Unlike retroviruses and LTR retrotransposons which undergo reverse transcription in cytoplasm, L1 RNA is reverse-transcribed in the nucleus19. L1 relies on a mechanism termed the Target Primed Reverse Transcription (TPRT) for retrotransposition20.Non-LTR retrotransposons are found throughout the eukaryotes, and examples of non-LTR retrotransposons include LINE-1  and SINE (Short Interspersed Nuclear Element). Human L1s account for ~17% of the genomic DNA with ~500,000 copies identified, and they represent the only autonomous transposable elements that are currently active in humans10. The retroelement suppression mechanisms include DNA methylation22, RNA interference24, and cellular DNA repair factors26. Also, studies suggest that several individual proteins are involved in L1 restriction. Examples of these proteins include: Three primer repair Exonuclease 1 (TREX1)27, Zinc finger Antiviral Protein (ZAP)28, RNA helicase Mov1029, RNaseL30, SAM domain and HD domain-containing protein 1(SAMHD1)32, and the APOBEC family of cytosine deaminases41.Despite the positive effects of retroelement mobilizations, such as contributing to genome evolution and gene diversity, excessive transposition events need to be suppressed, as several genetic diseases have been associated with L1-mediated insertional mutagenesis in germline and somatic cells43. The most ancient APOBEC family members include Activation Induced cytidine Deaminase (AID) and APOBEC2 (A2) proteins that are identified in jawless and cartilaginous fish44. AID initiates class-switch recombination and somatic hypermutation, two processes that occur during antibody maturation, by deaminating cytosine in single-stranded (ss)DNA to form promutagenic uracil. The ssDNA is available during transcription and initiates an error-prone DNA repair process that diversifies the antibody genes and enables a specialized recombination to occur for immunoglobulin class switching45. The A2 enzyme is involved in cardiac and skeletal muscle development47. APOBEC4 (A4) and APOBEC5 (A5) then emerged in amphibians, although their functions are yet to be identified49. During tetrapod evolution, duplication of AID led to the emergence of APOBEC1 (A1)50. A1 plays an important role in lipid transport by editing the apolipoprotein B mRNA in intestinal cells by introducing a uracil that results in a stop codon and truncated version of Apolipoprotein B that has a different function than the full length form51. A1 can also deaminate cytosine in ssDNA52. In placental mammals the APOBEC3 (A3) locus evolved and greatly expanded, which is thought to partly be due to the increasing number of retrotransposons in the genome43. While only one A3 gene is present in mice, up to seven A3 genes  are present in primates43. A3 genes have likely acquired the anti-L1 activities from the Activation Induced Cytidine Deaminase (AICDA)/A2-like gene from which they have evolved. Several studies have demonstrated that in a cell culture system that A3 family members could inhibit a number of mammalian L1s41. Since A3 genes are only limited to mammalian lineages, but L1s are far more ancient, dating back to the emergence of vertebrates, it was not surprising to find that APOBEC proteins, such as pre-mammalian AID and reptilian A1 from green anole lizard, are indeed capable of inhibiting L1 retrotransposition53.The APOBEC enzyme family emerged at the origin of vertebrates and have diversified throughout the vertebrate lineage57. For a deamination-dependent mechanism, during the reverse transcription of the L1 RNA, the (-)cDNA gets transiently exposed after RNA template degradation, which renders it susceptible to A3-mediated cytosine to uracil (C → U) deaminations57. The uracils present on L1 cDNA could trigger cDNA degradation through the actions of the host base excision repair pathway. Specifically, cellular uracil DNA N-glycosylase (UNG) excises the uracils from ssDNA, generating abasic sites and apurinic/apyrimidinic endonuclease (APE) produces nicks on the DNA which would induce L1 cDNA degradation. Alternatively, a uracil-containing cDNA may escape the degradation, become replicated to form a dsDNA, and integrate into the host chromosome, but be functionally inactivated through A3 induced C/G → T/A transition mutations that result from the polymerase using uracil as a template. For a deamination-independent mechanism of L1 restriction, the A3s can sequester L1 ribonucleoprotein (RNP) complexes to cytoplasmic compartments such as stress granules or physically interact with L1 RT and inhibit DNA polymerization during TPRT59. There is no clear agreement in the literature as to whether A3s utilize the deamination -dependent or -independent mode for L1 restriction. These ambiguities arose because no G → A mutations on the coding strand, which would indicate the occurrence of C → U deamination on the L1 cDNA, could be detected. However, none of these cell culture studies blocked the action of UNG60. Richardson et al., reasoned that the unsuccessful attempts to uncover A3-induced mutations were likely due to the degradation of uracil-containing cDNA intermediates by the UNG and APE-mediated DNA repair proteins57. In their study, using the Uracil Glycosylase Inhibitor (UGI) protein of Bacillus subtilis bacteriophage PBS161, they demonstrated that A3A restricted L1 through a deamination-dependent mechanism, suggesting that the mechanism by which A3 enzymes restricted L1 needed to be readdressed. These data also explained how mammalian L1 sequences analyzed on larger genome-wide scales could show more evidence of cytosine deamination and is in agreement with the known restriction mechanism of several endogenous retroviruses that show a G → A mutation bias65.A3s have been reported to restrict L1 by both deamination -dependent or -independent mechanisms70. Since haplotype II (hap II) and haplotype V (hap V) comprise the majority of the stable haplotypes we examined how they restricted L1 retrotransposition. Previous research has demonstrated that stable A3H could inhibit L1, however, a mechanism was not identified71. In this study, we demonstrate that both A3H hap II and hap V suppress L1 to a similar level but the inhibition of L1 does not involve DNA deamination, in contrast to A3A.To address whether the deamination-dependent mechanism of L1 inhibition is unique to A3A or common to other A3s, we examined how A3H restricted L1 retrotransposition in cell culture. There are at least seven A3H haplotypes that exist in humans. Three of the haplotypes are stable and catalytically active , three are thermodynamically unstable and have no discernable activity , and one has an intermediate stability and is catalytically active (I)54.To establish a context with which to interpret our results with A3H, we also included A3A and A3G in our study. These additional A3s were chosen based on previous publications indicating that A3A was a strong inhibitor of L1 retrotransposition and A3G was unable or weakly able to inhibit of L1 retrotransposition73  reporter cassette, which, when expressed, can convey resistance to G418. The neo reporter cassette has its own promoter and polyadenyation signal, and it consists of an antisense neo gene disrupted by an intron of the γ-globin gene, which is in the same transcriptional orientation as the L1 element  cells will only arise when a transcript initiated from the L1 promoter is spliced, reverse transcribed, its cDNA is re-inserted into the host chromosome, and the neo gene is transcribed from an internal promoter 38, can accommodate high levels of L1 retrotransposition events from ectopically expressed L1 reporter plasmids74 and do not express endogenous A3A, A3G and A3H proteins38.To study the influence of A3 proteins on L1 retrotransposition, a pre-established cell line based retrotransposition assay was performed73 Fig. . To deteent Fig. . Becauseter Fig. . The L1 R colonies were stained and counted. Transfection of L1 vector alone with no A3 expression plasmid generates a quantifiable amount of G418R colonies  and A3G  according to the previously published catalytic mutants78. In this experiment we tested only A3H hap II since both hap II and hap V restricted L1 retrotransposition similarly , although this has been observed under other experimental conditions81, and that indeed the mutations were acquired during integration of the L1 by TPRT, consistent with Richardson et al.57. Because A3A prefers to deaminate C → U within a 5′TC or 5′CC context (underlined C is deaminated)83, we determined whether these G → A mutations on the genomic strand occurred within the preferred deamination motif as a confirmation of A3A deamination activity. Deamination of these motifs in the cDNA will result in 5′GA → 5′AA or 5′GG → AG mutations in the coding strand. Out of the 16 G → A mutations we recovered in the neo gene, two mutations were within the 5′GA → 5′AA context and 1 mutation was within the 5′GG → AG context (data not shown). In addition, other mutations at 5′GC → 5′AC and 5′GT → 5′AT motifs were observed that may or may not be due to A3A catalytic activity (data not shown). However, in the presence of UGI, the majority of the G → A mutations occurred within the 5′GA → 5′AA context, confirming that UGI blocked removal of uracils formed by A3A catalytic activity 66. A3H hap II and hap V also did not appear to be able to deaminate L1 mRNA, since no C → T mutations with the correct sequence context were found cDNA or physically impair RT’s activity86. Based on these observations and that both A3H Hap II and Hap V are primarily cytoplasmic proteins67, we hypothesize A3H could inhibit L1 mobility by binding to the L1 RNP in the cytoplasm, similar to what has been reported for A3C and A3D59.We found A3H hap II and hap V are able to suppress L1 retrotransposition to a moderate level and there were no apparent differences in their L1 restriction abilities Fig. . Three ld84 Fig. . However 85 Fig. . CytoplaThe analysis of the L1 restriction ability of A3A, A3G, and A3H hap II and hap V have demonstrated that each of the A3s has a unique effect on L1. In particular, although L1 restriction by A3s can be deamination-dependent, as shown for A3A, this does not appear to be applicable to all A3 enzymes. These data suggest that reanalysis of A3-mediated L1 restriction in the presence of UGI for all A3 family members is warranted to fully understand their mechanism of activity.88 and were cloned into pcDNA6-V5/His.A using XbaI and XhoI cloning sites giving the enzymes a C-terminal V5-His fusion tag. Mutants of A3 enzymes  were made using the wild type construct for site-directed mutagenesis . The Uracil DNA glycosylase inhibitor gene was synthesized by GenScript with codon optimization for human cells and subcloned into pcDNA6, but was expressed without the tags for the experiments or subcloned into pcDNA3.1 with a C-terminal V5 tag for checking expression by immunoblotting. All constructed plasmids were verified by sequencing.L1 expression plasmid pJM101/L1.3-Neo was kindly provided by Dr. John V. Moran (University of Michigan). The cDNA of A3A, A3G and A3H have been previously reported5 cells/well in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS. Approximately 22 h after plating, Hela cells were co-transfected with 1 μg of L1 retrotransposon encoding plasmid (pJM101/L1.3) and 1 μg of an A3 encoding plasmid or empty vector. Transfections were performed using GeneJuice (EMD Millipore) according to the manufacturer’s protocol. Media was replaced the following day. Approximately 48 h post-transfection, cells were re-seeded into 6-well plates (at 1 in 5 dilution) and the next day attached cells were subjected to selection with 400 μg/mL G418 (Life Technologies). During the reseeding process, cells were checked for viability by trypan blue staining. We consistently observed a cell viability > 95% indicating that expression of A3A, A3G, or A3H did not cause cellular toxicity. G418 selection was carried out for 11 days and the selection media were replaced every other day. Afterwards, G418R colonies were washed twice with PBS, fixed with 2% paraformaldehyde/0.4% glutaraldehyde, and stained with 0.1% crystal violet solution.HeLa cells were seeded in each well of a 6-well tissue culture plate at a density of 2 × 10To detect A3 expression levels, cells were washed in 1x PBS, harvested in Laemmli sample buffer (58 mM Tris (pH 6.8), 5% glycerol, 2% sodium dodecyl sulphate (SDS), 1.5% dithiothreitol (DTT)), and 40 μg of total protein was resolved by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and A3 or UGI in the cell lysates were probed with primary antibodies  and rabbit monoclonal α-tubulin, 1:1000 (Sigma)). After incubation with the secondary antibodies  the tagged proteins and loading control was detected simultaneously on the same membrane by using the Licor/Odyssey system.neo gene using the forward primer (G418Neoseq For) 5′-TCGGGAGCGGCGATACCGT-3′ and the reverse primer (G418Neoseq Rev) 5′-CGGTGCCCTGAATGAGCTTCA-3′ with the Q5 High-Fidelity polymerase (New England Biolabs). The PCR cycle used had an initial denaturation step at 98 °C (3 min) followed by 35 cycles of amplification . PCR products were electrophoresed to separate unspliced (of plasmid origin) and spliced (of integrated L1 origin) PCR products. The spliced product was gel purified with the GenElute Gel Extraction kit (Sigma) and cloned into pJET1.2/blunt vector . After transformation into Escherichia coli DH5α cells, clones were picked and sent for sequencing using a kit-specific primer (pJET1.2 Forward) at the National Research Council DNA sequencing Facility . Sequence alignments were performed using Clustal Omega.Hela cells were co-transfected with 1 μg of A3 and L1 plasmids in the presence or absence of 1 μg UGI vector as described above. Cellular DNA from the transfected cells was extracted 72 hr post-transfection using DNAzol reagent (Thermo Fisher). Extracted DNA was used as a template for PCR amplification of the spliced E. coli (New England Biolabs) was used as a control. For DNA visualization the samples were mixed with an equal volume of formamide/EDTA loading dye, resolved on a urea denaturing 16% v/v polyacrylamide gel, and scanned using a Typhoon Trio multipurpose scanner .HeLa cells were transfected with 1 μg of L1 retrotransposon encoding plasmid (pJM101/L1.3) and either 1 μg empty vector or UGI vector as described above. Cells were harvested and lysed 72 h after the transfection in buffer containing 50 mM Tris-Cl pH 7.4, 1% Nonidet-P40, 0.1% sodium deoxycholate, 10% glycerol, 150 mM NaCl, 1 mM DTT, 20 μg/mL RNaseA and EDTA-free protease inhibitor (Roche). To test for UGI activity, 8 μL of the lysate was incubated with 100 nM of a 44 nt ssDNA oligonucleotide with a single deoxyuridine and a 5′ Fluorescein (5′ (Fluorescein)-AAA GAG AAA GTG ATA AAC AAA GAG TAA AGU AGA TAG AGA GTG ATA 3′) for 15 min at 37 °C. To cleave the DNA at abasic sites generated by the action of UNG, the DNA was then heated at 95 °C for 10 min in the presence of 0.2 M NaOH. Purified UDG from No datasets were generated or analyzed during the current study.