[Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter].

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3.

Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability.

Integrating RNA-seq and ChIP-seq data to characterize long non-coding RNAs in Drosophila melanogaster.

BACKGROUND: Recent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data (H3K4me3, H3K36me3 and Pol II) to detect signatures of active transcription for reported lncRNAs. RESULTS: We have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the poly(A)-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a poly(A) tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24% of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not. CONCLUSIONS: In this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets (93 sets in total) from multiple sources (lncRNAs, RNA-seq and ChIP-seq). The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.

Rapid Osseointegration of Titanium Implant With Innovative Nanoporous Surface Modification: Animal Model and Clinical Trial.

OBJECTIVES: SLAffinity is the hybrid topography consisting of micropits and nanoporous TiO2 layers through electrochemical oxidation to mimic the natural bony environment. The aim of this study was to examine the rate of osseointegration in animal models and to further investigate the stability for implants with SLAffinity-treated surface in the clinical trial. MATERIALS AND METHODS: Implants were installed in the mandibular canine-premolar area of 12 miniature pigs. Each pig received 2 implants with the same shapes but with different chemical surfaces. In the clinical trial, 25 patients were included. Each patient received 1 SLAffinity-treated implant on the posterior area of either arch. Resonance frequency analysis and computed tomography were assessed weekly over the first 12 weeks after implant placement. RESULTS: The results found that surface treatment did affect the bone-to-implant contact (BIC) significantly. Comparison of BIC at 3 weeks in animal study showed that the SLAffinity-treated implants presented significantly higher values than machine surface implants. SLAffinity-treated implants also proved clinically successful through 12 months, ready for prosthodontic restoration. CONCLUSION: The effect of SLAffinity treatments enhanced osseointegration significantly, especially at early stages of bone healing. Clinical trial finding, furthermore, ensured that the SLAffinity treatment was a reliable surface modification alternative.

Clinical treatment outcomes for 40 patients with bisphosphonates-related osteonecrosis of the jaws.

BACKGROUND/PURPOSE: Bisphosphonates (BPs) are used to treat osteoporosis and bone metastases from malignancy. They may result in BPs-related osteonecrosis of the jaws (BRONJ) in a subset of patients receiving BPs. This study examined whether conservative or aggressive surgical approach could result in successful treatment of BRONJ lesions and assessed whether concomitant steroid administration or tobacco smoking habit might hinder the remission of BRONJ lesions. METHODS: The 40 BRONJ patients were evenly divided into four different groups. Group 1 contained 10 patients with concomitant corticosteroid medication but without smoking habit. Group 2 contained 10 patients with smoking habit but without concomitant corticosteroid medication. Groups 3 and 4 each consisted of 10 patients without concomitant corticosteroid medication and smoking habit. To avoid bias, each group contained equal number of patients with different stages of BRONJ. Patients in Groups 1, 2, and 3 received conservative treatment, including antibiotic coverage, antibacterial solution irrigation, and minor surgical debridement. Patients in Group 4 were treated with aggressive surgical excision of necrotic bone segment. RESULTS: The mean duration to achieve complete remission of BRONJ lesion was 19.7±0.6, 18.2±0.5, 13.0±1.0, and 7.6±1.1 months for patients in Groups 1, 2, 3 and 4, respectively. Student's t-test showed significant differences in the mean duration to achieve complete remission of BRONJ lesion between Groups 1 and 3, between Groups 2 and 3, between Groups 3 and 4, between Groups 1 and 4, and between Groups 2 and 4 (all p values < 0.001). CONCLUSION: Although both conservative and aggressive surgical approaches can result in successful treatment of BRONJ lesions, aggressive surgical treatment needs a shorter mean duration to achieve complete remission of BRONJ lesion than conservative treatment. Concomitant corticosteroid administration or tobacco smoking may prolong the duration for complete remission of BRONJ lesion.

Antibacterial nanostructured composite films for biomedical applications: microstructural characteristics, biocompatibility, and antibacterial mechanisms.

Hydrogenated Cu-incorporated diamond-like carbon (a-C:H/Cu) films were prepared in the present study using a radio-frequency plasma magnetron sputtering system at various CH4/Ar gas ratios. The a-C:H/Cu films were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, transmission electron microscopy, nano-indentation and a contact angle goniometer. The antibacterial properties and cell cytotoxicity of a-C:H/Cu films were evaluated as per JIS Z2801:2010 and ISO 10993-5 specifications, respectively. The analytical results revealed that the production of a-C:H/Cu films varied with the CH4/Ar ratio, and the phase transformation (amorphous-like → nano-polycrystalline structure) was induced by Cu doping/ion bombardment and radical reactions. Moreover, it was found that the microhardness of the a-C:H/Cu films decreased with increasing Ar fraction in the gas ratio. The a-C:H/Cu films exhibited a high hydrophobic surface feature. The film which contained 77.3 ± 4.4 at.% Cu did not influence cell adhesion and proliferation behaviors. Antibacterial tests also demonstrated that a-C:H/Cu films possessed excellent antibacterial properties. Therefore, a-C:H/Cu films could be developed as promising antibacterial coatings for biomedical applications.

Blood responses to titanium surface with TiO2 nano-mesh structure.

OBJECTIVES: The goal of this study was to enhance the blood responses to titanium (Ti) surfaces used for dental implant application through the formation of a TiO2 nano-mesh surface layer produced by a fast electrochemical anodization treatment. MATERIAL AND METHODS: Electrochemical anodization treatments with different anodization currents and temperatures in an alkaline solution were used to create a nano-mesh oxide layer on polished Ti surface. Surface characterizations of the mesh structure were carried out using thin-film X-ray diffractometer, field-emission scanning electron microscope, and atomic force microscope. The blood responses, including the blood-clotting ability and platelet adhesion morphology, to the test Ti surfaces were evaluated. The blood-clotting ability, in terms of optical density of blood, was statistically analyzed using a nonparametric method, Kruskal-Wallis test, for the factor of anodization treatment. RESULTS: A multilayer TiO2 nano-mesh structure was rapidly formed on the polished Ti surface using a simple electrochemical anodization treatment in an alkaline solution. The TiO2 nano-mesh had an average mesh size between 34 and 93 nm, depending on the anodization current and temperature. The features on the TiO2 nano-mesh structure on the anodized Ti surface were of a similar size scale as blood proteins, giving the material better blood clot ability (P<0.05) and improved platelet activation and aggregation as compared with an untreated polished Ti surface. CONCLUSIONS: The formation of TiO2 nano-mesh on the Ti surfaces was shown to enhance blood responses, which we expect to promote cell growth in the application of dental implants.

Curcumin upregulates insulin-like growth factor binding protein-5 (IGFBP-5) and C/EBPalpha during oral cancer suppression.

Curcumin is a common food ingredient derived from the plant Curcuma longa and is a potent drug against tumorigenesis. Both insulin-like growth factor binding protein-5 (IGFBP-5) and CCAAT/enhancer-binding protein alpha (C/EBPalpha) are suppressors of head and neck carcinogenesis. We identified curcumin as an inducer of IGFBP-5 expression in multiple types of oral keratinocytes; furthermore, curcumin induces IGFBP-5 promoter activity in SAS oral cancer cells. Promoter deletion mapping identified a region (nt -71 to nt -59 relative to the transcription start site) as containing a C/EBPalpha-binding element that is indispensable for curcumin-mediated IGFBP-5 upregulation. Chromatin immunoprecipitation assays revealed that in vivo binding of C/EBPalpha to this region was remarkably increased in the presence of curcumin. Curcumin increased nuclear C/EBPalpha expression and IGFBP-5 expression through p38 activation and this was abrogated by SB203580 treatment. Furthermore, MKK6 expression activated p38 and C/EBPalpha, increasing IGFBP-5 promoter activity and expression. Finally, curcumin-induced IGFBP-5 expression is associated with the suppression of xenograft tumorigenesis in mice due to oral cancer cells. We conclude that curcumin activates p38, which, in turn, activates the C/EBPalpha transactivator by interacting with binding elements in the IGFBP-5 promoter. The consequential upregulation of C/EBPalpha and IGFBP-5 by curcumin is crucial to the suppression of oral carcinogenesis.

Duration for apical barrier formation in necrotic immature permanent incisors treated with calcium hydroxide apexification using ultrasonic or hand filing.

BACKGROUND/PURPOSE: Traumatic injury usually results in pulp necrosis of immature permanent incisors in children aged 7-10 years. Calcium hydroxide apexification is the most common treatment for necrotic, immature permanent teeth. This study compared the duration for apical barrier formation in necrotic immature permanent incisors treated with calcium hydroxide apexification using ultrasonic or hand filing. METHODS: Thirty-two trauma-induced necrotic immature permanent incisors with or without a periapical lesion (PL) were selected from children aged 7-10 years. They were evenly divided into four groups. Teeth in groups 1 (with PL) and 2 (without PL) were treated with ultrasonic filing, and teeth in groups 3 (with PL) and 4 (without PL) were treated with hand filing. The canals were cleaned with 0.2% chlorhexidine solution during treatment and then compactly filled with calcium hydroxide. The patients were followed up once every 1-3 weeks to change the intracanal medication and to detect when the apical barrier formed. RESULTS: The mean duration for apical barrier formation was 11.1 +/- 1.1 weeks, 11.8 +/- 1.0 weeks, 13.3+/-0.9 weeks and 13.4 +/- 0.7 weeks for groups 1, 2, 3 and 4, respectively. Student's t test showed significant differences in the mean duration for apical barrier formation between groups 1+2 and 3 + 4 (p = 0.000), groups 1 and 3 (p = 0.000), and groups 2 and 4 (p = 0.002). These results indicated that teeth treated with ultrasonic filing required a shorter mean duration for apical barrier formation than teeth treated with hand filing regardless of the presence of PL or not. CONCLUSION: Ultrasonic filing with 0.2% chlorhexidine as an irrigant is effective for disinfection of the root canal and can shorten the duration for apical barrier formation in necrotic permanent incisors treated with calcium hydroxide apexification.

Corrosion resistance of different nickel-titanium archwires in acidic fluoride-containing artificial saliva.

OBJECTIVE: To test the hypothesis that different nickel-titanium (NiTi) archwires may have dissimilar corrosion resistance in a fluoride-containing oral environment. MATERIALS AND METHODS: Linear polarization test, a fast electrochemical technique, was used to evaluate the corrosion resistance, in terms of polarization resistance (R(p)), of four different commercial NiTi archwires in artificial saliva (pH 6.5) with various NaF concentrations (0%, 0.01%, 0.1%, 0.25%, and 0.5%). Two-way analysis of variance was used to analyze R(p) with the factors of archwire manufacturer and NaF concentration. Surface characterizations of archwires were analyzed using scanning electron microscopy, atomic force microscopy, and x-ray photoelectron spectroscopy. RESULTS: Both archwire manufacturer and NaF concentration had a significant influence on R(p) of NiTi archwires. Different surface topography was present on the test NiTi archwires that contained the similar surface chemical structure (TiO(2) and trace NiO). The surface topography did not correspond to the difference in corrosion resistance of the NiTi archwires. Increasing the NaF concentration in artificial saliva resulted in a decrease in R(p), or corrosion resistance, of all test NiTi archwires. The NiTi archwires severely corroded and showed similar corrosion resistance in 0.5% NaF-containing environment. CONCLUSIONS: Different NiTi archwires had dissimilar corrosion resistance in acidic fluoride-containing artificial saliva, which did not correspond to the variation in the surface topography of the archwires. The presence of fluoride in artificial saliva was detrimental to the corrosion resistance of the test NiTi archwires, especially at a 0.5% NaF concentration.

Areca nut extract-treated gingival fibroblasts modulate the invasiveness of polymorphonuclear leukocytes via the production of MMP-2.

BACKGROUND: Areca nut chewing is associated with an increase in the incidence of oral neoplastic or inflammatory diseases. Aberrations in matrix metalloprotease (MMP) expression are associated with the pathogenesis of oral diseases. This study investigated the potential effects of areca nut extract (ANE) on human gingival fibroblasts and the consequential impacts on inflammatory pathogenesis. METHODS: Analyses of senescence marker, cell viability, changes of the cell cycle, and cell granularity in gingival fibroblasts together with an assessment of the invasiveness of polymorphonuclear (PMN) leukocytes after treatment with the supernatant of ANE-treated gingival fibroblasts were performed to characterize the phenotypic impacts. Western blotting and gelatin zymography were used to assay the expression and activity of MMP-2. RESULTS: Chronic subtoxic (<10 microg/ml) ANE treatment resulted in premature growth arrest, appearance of senescence-associated beta-galactosidase activity and various other senescence-associated phenotypes in gingival fibroblasts. Gingival fibroblasts established from older individuals had a higher propensity to become ANE-induced senescent gingival fibroblasts. An activation of MMP-2 was identified in senescent cells. PMN leukocytes treated with the supernatant of ANE-induced senescent cells exhibited a significant increase in invasiveness, which was abrogated by both a MMP-2 blocker and a MMP-2 nullifying antibody. CONCLUSIONS: This study provides evidence whereby MMP-2 secreted from ANE-induced senescent gingival fibroblasts would facilitate the invasiveness of PMN leukocytes, which could be associated with the oral inflammatory process in areca chewers.

Fracture resistance of Nd:YAG laser-welded cast titanium joints with various clinical thicknesses and welding pulse energies.

The purpose of this study was to evaluate the fracture resistance of Nd:YAG laser-welded cast titanium (Ti) joints with various clinical thicknesses and welding pulse energies. A four-point bending test was used to assess the effects of various specimen thicknesses (1-3 mm) and welding pulse energies (11-24 J) on the fracture resistance of Nd:YAG laser-welded Ti dental joints. Fracture resistance was evaluated in terms of the ratio of the number of fractured specimens to the number of tested specimens. As for the fracture frequencies, they were compared using the Cochran-Mantel-Haenszel test. Morphology of the fractured Ti joints was observed using a scanning electron microscope. Results showed that decreasing the specimen thickness and/or increasing the welding pulse energy, i.e., increasing the welded area percentage, resulted in an increase in the fracture resistance of the Ti joint. Where fracture occurred, the fracture site would be at the center of the weld metal.

Formation of TiO(2) nano-network on titanium surface increases the human cell growth.

OBJECTIVES: This study was to improve human cell growth on titanium (Ti) used for dental implants through formation of a nano-network surface oxide layer created by an electrochemical anodization treatment. METHODS: An electrochemical anodization treatment was used to produce a network oxide layer on Ti surface. Surface characterization of the network layer was carried out using thin film X-ray diffractometer and field emission scanning electron microscopy. Human bone marrow mesenchymal stem cells (hMSCs) were made to express green fluorescent protein (GFP) by retroviral transduction. The GFP signal was measured in situ to assess in vitro and in vivo cell growth on Ti surfaces. In vivo experiments on Ti-supported cell growth were carried out on the back skin of nude mice. Alizarin red staining and immunofluorescent staining were used to observe cell differentiation. RESULTS: A multilayer TiO(2) nano-network was produced rapidly on Ti surface using a simple electrochemical anodization treatment. The TiO(2) nano-network layer on the anodized Ti surfaces significantly improved in vitro and in vivo hMSC growth, as assessed by measurement of GFP fluorescence, relative to hMSC growth on untreated Ti surface. The TiO(2) nano-network layer on the anodized Ti surfaces can also induce the differentiation of hMSCs after 28-day in vivo test. SIGNIFICANCE: The formation of TiO(2) nano-network on the Ti surfaces can increase the hMSC growth in vitro and in vivo.