Detection of CpG methylation level using methyl-CpG-binding domain-fused fluorescent protein.

Methylation of cytosine to 5-methylcytosine on CpG dinucleotides is the most frequently studied epigenetic modification involved in the regulation of gene expression. In normal tissues, tissue-specific CpG methylation patterns are established during development. In contrast, alterations in methylation patterns have been observed in abnormal cells, such as cancer cells. Cancer type-specific CpG methylation patterns have been identified and used as biomarkers for cancer diagnosis. In this study, we developed a hybridization-based CpG methylation level sensing system using a methyl-CpG-binding domain (MBD)-fused fluorescent protein. In this system, the target DNA is captured by a complementary methylated probe DNA. When the target DNA is methylated, a symmetrically methylated CpG is formed in the double-stranded DNA. MBD specifically recognizes symmetrical methyl-CpG on double-stranded DNA; therefore, the methylation level is quantified by measuring the fluorescence intensity of the bound MBD-fused fluorescent protein. We prepared MBD-fused AcGFP1 and quantified the CpG methylation levels of the target DNA against SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) using MBD-AcGFP1. This detection principle can be applied to the simultaneous and genome-wide modified base detection systems using microarrays coupled with modified base binding proteins fused to fluorescent proteins.

Hybridization-based CpG methylation level detection using methyl-CpG-binding domain-fused luciferase.

Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation level detection method using methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc). In this method, methylated probe oligonucleotides were used to capture target oligonucleotides. Fully methylated and hemimethylated double-stranded DNA (dsDNA) was formed by hybridization of the methylated captured oligonucleotides with methylated or unmethylated target oligonucleotides, respectively. MBD-Fluc specifically binds to fully methylated dsDNA but not to hemimethylated dsDNA; therefore, methylated target oligonucleotides can be detected by measuring the luciferase activity of the bound MBD-Fluc. Using the corresponding methylated probe oligonucleotides, the CpG methylation levels of SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) oligonucleotides were quantified. Moreover, we demonstrated that the emission detection signal was not affected by the methylation state of the overhang region of the target oligonucleotide, which was not hybridized to the probe oligonucleotide, indicating that methylated CpG of the target region could be accurately detected. Unmethylated-CpG-binding domain-fused luciferases and 5-hydroxymethyl-CpG-binding domain-fused luciferases have been constructed, suggesting that other modified bases can be detected by the same platform.

Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer.

Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (Fluc) and unmethyl-CpG binding domain (CXXC)-fused Oplophorus luciferase (Oluc). Moreover, BRET-based hydroxymethylation and hemi-methylation level sensing systems have been developed using hydroxymethyl-CpG and hemi-methyl-CpG binding domain-fused Fluc. These studies suggest that target epigenetic modifications can be simultaneously quantified using target-modification-binding protein-fused luciferases. In this study, we focused on the SnoopTag (SnT)/SnoopCatcher (SnC) protein ligation system to establish a universal design for fusion protein construction for any combination. SnT spontaneously forms an isopeptide bond with SnC; therefore, any kind of fusion protein would be constructed by the SnT/SnC system. To establish the proof of concept, MBD-SnT, CXXC-SnT, and SnC-Oluc were prepared and ligated MBD-SnT or CXXC-SnT to SnC-Oluc. The ligation products of MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc showed luciferase activity and specific binding activity to methyl-CpG and unmethyl-CpG, respectively. The BRET signal using MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc increased the amount of methyl-CpG and unmethyl-CpG in genomic DNA, respectively. There was a significant negative correlation between the BRET signals; therefore, the global DNA methylation level was quantified using the BRET signals (R2 = 0.99, and R.S.D. <3.5%). These results indicate that the SnT/SnC protein ligation system can be utilized to construct target modification-binding protein-fused luciferases in any combination that detects target modifications in genomic DNA based on BRET.

Quantitative detection of CpG methylation level on G-quadruplex and i-motif-forming DNA by recombinase polymerase amplification.

Detection of CpG methylation levels holds immense potential for application in medical diagnosis of various diseases. In this study, we report the development of a recombinase polymerase amplification (RPA)-based CpG methylation level sensing system on G-quadruplex (G4) and intercalated motif (i-motif)-forming regions, which are stabilized by CpG methylation. This detection system is based on the principle that DNA polymerase is stalled at the methylated G4 and i-motif-forming region, which results in a decrease in the initial elongation efficiency of RPA. This reduction in turn affects the onset of amplification depending on the extent of CpG methylation; therefore, the methylation level is quantified by RPA. We demonstrate that the onset of amplification was delayed by CpG methylation when PCR products containing the vascular endothelial growth factor (VEGF) G4 and i-motif-forming region were used as the template. Furthermore, onset of amplification was delayed with the increase in CpG methylation of the VEGF region on genomic DNA. These results demonstrate that the sensing system is capable of directly detecting the methylation level at a constant temperature (39 °C) within 30 min without performing bisulfite conversion or affinity capture of methylated DNA.

Bioluminescence Resonance Energy Transfer for Global DNA Methylation Quantification.

Global hypomethylation of genomic DNA is associated with genomic instability and carcinogenic processes. The loss of DNA methylation has been reported in several cancers; therefore, global methylation levels have been considered as biomarkers for cancer diagnosis. Bisulfite conversion analysis has been widely used as the gold standard method for quantification of DNA methylation levels. However, this method requires cumbersome and time-consuming steps. To quantify global DNA methylation levels in homogeneous solutions, we exemplify a sensing system based on bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (MBD-FLuc) and unmethyl-CpG binding domain (CXXC)-fused firefly luciferase (CXXC-FLuc). MBD-FLuc and CXXC-FLuc bind to methylated and unmethylated CpGs, respectively, in the genomic DNA to excite BOBO-3, an intercalating dye on genomic DNA. These BOBO-3 emission intensities depend on the methylated and unmethylated CpG content. The global DNA methylation levels can be quantified from the BOBO-3 emission intensities. Moreover, we introduce a multicolor BRET assay using MBD-FLuc and CXXC-fused Oplophorus luciferase (CXXC-OLuc) for the simultaneous quantification of methylated and unmethylated CpG content in genomic DNA. CXXC-OLuc excites the BOBO-1 DNA-intercalating dye depending on the unmethylated CpG content. Thus, the emission intensities of BOBO-1 and BOBO-3 excited by CXXC-OLuc and MBD-FLuc, respectively, can be simultaneously measured, thereby enabling the determination of global DNA methylation level in a single step. Here, we describe the detailed protocols for the expression of MBD-FLuc, CXXC-FLuc, and CXXC-OLuc in Escherichia coli and determine the global DNA methylation levels using these BRET assays.

Quantification of Global DNA Hydroxymethylation Level Using UHRF2 SRA-Luciferase Based on Bioluminescence Resonance Energy Transfer.

5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD (R2 = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng µL-1. In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.

Investigating the Effects of Dehydrated Human Amnion-Chorion Membrane on Periodontal Healing.

Each growth factor (GF) has different effects and targets, and plays a critical role in periodontal healing. Dehydrated human amnion-chorion membrane (dHACM) contains various GFs and has been used to enhance wound healing. The purpose of this study was to evaluate the effects of dHACM on periodontal healing, using in vitro and in vivo experimental approaches. Standardized periodontal defects were created in rats. The defects were randomly divided into three groups: Unfilled, filled with hydroxypropyl cellulose (HPC), and dHACM+HPC. At 2 and 4 weeks postoperatively, periodontal healing was analyzed by microcomputed tomography (micro-CT), and histological and immunohistochemical analyses. In vitro, periodontal ligament-derived cells (PDLCs) isolated from rat incisors were incubated with dHACM extract. Cell proliferation and migration were evaluated by WST-1 and wound healing assay. In vivo, micro-CT examination at 2 weeks revealed enhanced formation of new bone in the dHACM+HPC group. At 4 weeks, the proportions of vascular endothelial growth factor (VEGF)-positive cells and α-smooth muscle actin (α-SMA)-positive blood vessels in the dHACM+HPC group were significantly greater than those in the Unfilled group. In vitro, dHACM extracts at 100 µg/mL significantly increased cell proliferation and migration compared with control. These findings suggest that GFs contained in dHACM promote proliferation and migration of PDLCs and angiogenesis, which lead to enhanced periodontal healing.

Destabilization of DNA and RNA G-quadruplex structures formed by GGA repeat due to N6-methyladenine modification.

N6-methyladenine (m6A) is the most abundant RNA modification in eukaryotic RNA. Further, m6A has been identified in the genomic DNA of both eukaryotes and prokaryotes. The G-quadruplex (G4) structure is a non-canonical nucleic acid structure formed by the stacking of G:G:G:G tetrads. In this study, we evaluated the effect of m6A modifications on G4 structures formed by GGA repeat oligonucleotides, d(GGA)8, d(GGA)4, and r(GGA)4. The d(GGA)8 forms an intramolecular tetrad:heptad:heptad:tetrad G4 structure, while d(GGA)4 forms a dimerized intermolecular tetrad:heptad:heptad:tetrad G4 structure. r(GGA)4 forms a dimerized intermolecular tetrad:hexad:hexad:tetrad G4 structure. Circular dichroism melting analysis demonstrated that (1) m6A modifications destabilized the G4 structure formed by d(GGA)8, (2) m6A modification at A3 disrupted the G4 structure formed by d(GGA)4, and (3) m6A modification at A3 destabilized the G4 structure formed by r(GGA)4. m6A modifications may be involved in controlling G4 structure formation to regulate biological functions.

Stabilization of VEGF i-motif structure by CpG methylation.

The intercalated motif (i-motif) is a non-canonical nucleic acid structure formed by intercalated hemi-protonated cytosine base pairs (C-C+) under acidic conditions. The i-motif structure formation is involved in biological processes such as transcription regulation. Therefore, the identification of factors controlling i-motif formation is important in elucidating the cellular functions it controls. We previously reported that the VEGF G-quadruplex structure is stabilized by CpG methylation. In this study, the effect of CpG methylation on the stability of the VEGF i-motif structure was investigated. The VEGF i-motif-forming oligonucleotide contains four cytosines on CpG sites, and three of the four cytosines (C4, C15, and C20) are involved in C-C+ formation in the i-motif structure. Circular dichroism (CD) spectra analysis demonstrated that full CpG methylation increased the pH of mid transition (pHT) of the i-motif structure by 0.1, and the melting temperature (Tm) by 5.1 °C in 25 mM sodium cacodylate buffer at pH 5.0. Moreover, single methylation at C4, C15, and C20 increased Tm by 0.5, 1.7, and 2.0 °C in the buffer, respectively. These results demonstrated that CpG methylation stabilized the VEGF i-motif structure.

Thermal Stability Changes in Telomeric G-Quadruplex Structures Due to N6-Methyladenine Modification.

N6-methyladenine modification (m6dA) has recently been identified in eukaryote genomic DNA. The methylation destabilizes the duplex structure when the adenine forms a Watson-Crick base pair, whereas the methylation on a terminal unpaired adenine stabilizes the duplex structure by increasing the stacking interaction. In this study, the effects of m6dA modification on the thermal stability of four distinct telomeric G-quadruplex (G4) structures were investigated. The m6dA-modified telomeric oligonucleotide d[AGGG(TTAGGG)3] that forms a basket-type G4 in Na+, d[(TTAGGG)4TT] that forms a hybrid-type G4 in K+ (Form-2), d[AAAGGG(TTAGGG)3AA] that forms a hybrid-type G4 in K+ (Form-1), and d[GGG(TTAGGG)3T] that forms a basket-type G4 with two G-tetrads in K+ (Form-3) were analyzed. Circular dichroism melting analysis demonstrated that (1) A7- and A19-methylation destabilized the basket-type G4 structure that formed in Na+, whereas A13-methylation stabilized the structure; (2) A15-methylation stabilized the Form-2 G4 structure; (3) A15- and A21-methylations stabilized the Form-1 G4 structure; and (4) A12-methylation stabilized the Form-3 G4 structure. These results suggest that m6dA modifications may affect the thermal stability of human telomeric G4 structures in regulating the biological functions.

Healing of Experimental Periodontal Defects Following Treatment with Fibroblast Growth Factor-2 and Deproteinized Bovine Bone Mineral.

The aim of this study was to investigate the effects of fibroblast growth factor (FGF)-2 used in combination with deproteinized bovine bone mineral (DBBM) on the healing of experimental periodontal defects. Periodontal defects created in rats were treated by FGF-2, DBBM, FGF-2 + DBBM, or left unfilled. Microcomputed tomography, histological, and immunohistochemical examinations were used to evaluate healing. In vitro cell viability/proliferation on DBBM with/without FGF-2 was assessed by WST-1. Cell behavior was analyzed using scanning electron and confocal laser scanning microscopy. Osteogenic differentiation was evaluated by staining with alkaline phosphatase and alizarin red. Bone volume fraction was significantly greater in FGF-2 and FGF-2 + DBBM groups than in other groups at 2 and 4 weeks postoperatively. In histological assessment, newly formed bone in FGF-2 and FGF-2 + DBBM groups appeared to be greater than other groups. Significantly greater levels of proliferating cell nuclear antigen-, vascular endothelial growth factor-, and osterix-positive cells were observed in FGF-2 and FGF-2 + DBBM groups compared to Unfilled group. In vitro, addition of FGF-2 to DBBM promoted cell viability/proliferation, attachment/spreading, and osteogenic differentiation. The combination therapy using FGF-2 and DBBM was similarly effective as FGF-2 alone in the healing of experimental periodontal defects. In certain bone defect configurations, the combined use of FGF-2 and DBBM may enhance healing via promotion of cell proliferation, angiogenesis, and osteogenic differentiation.

Theoretical Study on Third-Order Nonlinear Optical Properties for One-Hole-Doped Diradicaloids.

We investigate the relationships between open-shell character and longitudinal static second hyperpolarizabilities γ for one-hole-doped diradicaloids using the strong-correlated ab initio molecular orbital methods and simple one-dimensional (1D) three-site two-electron (3s-2e) models. As examples of one-hole-doped diradicaloids, we examine H3 +, methyl radical trimer cation ((CH3)3 +), silyl radical trimer cation ((SiH3)3 +), and 1,2,3,5-dithiadizolyl trimer cation (DTDA3 +). For H3 +, the static γ exhibits negative values and shows a monotonic increase in amplitude with an increase in the open-shell character defined by a neighbor-site interaction (y S). On the other hand, it is found for (CH3)3 +, (SiH3)3 +, and DTDA3 + that the static γ value exhibits similar behavior to that for H3 + up to an intermediate y S value, while it takes the negative maximum at a large y S value, followed by a decrease in γ amplitude, and subsequently, γ changes to positive values with a drastic increase for larger y S values. For example, in DTDA3 +, the negative/positive γ values, -69 × 105/700 × 105 au at y S = 0.75/0.87, exhibit significant enhancements in amplitude, 2.4/24 times as large as that (-29 × 105 au) at intermediate y S = 0.59 as is often the case in DTDA2. Using the 1D 3s-2e valence-bond configuration interaction model, these sign inversions and drastic increase in the amplitude of γ are found to originate in the differences in Coulomb interactions between valence electrons, between valence and core electrons, and between valence electrons and nuclei. These results contribute to pave the way for the construction of novel control guidelines for the amplitude and sign of γ for one-hole-doped diradicaloids.

Periodontal surgery using rhFGF-2 with deproteinized bovine bone mineral or rhFGF-2 alone: 2-year follow-up of a randomized controlled trial.

AIM: To compare outcomes of rhFGF-2 + DBBM therapy with rhFGF-2 alone in the treatment of intrabony defects. This study provides 2-year follow-up results from the previous randomized controlled trial. MATERIALS AND METHODS: Defects were randomly allocated to receive rhFGF-2 + DBBM (test) or rhFGF-2 (control). Treated sites were re-evaluated at 2 years postoperatively, using original clinical and patient-centred measures. RESULTS: Thirty-eight sites were available for re-evaluation. At 2 years, both groups showed a significant improvement in clinical attachment level (CAL) from baseline. A gain in CAL of 3.4 ± 1.3 mm in the test group and 3.1 ± 1.5 mm in the control group was found. No significant inter-group difference was noted. Both groups showed a progressive increase in radiographic bone fill (RBF). The test treatment yielded greater RBF (56%) compared with the control group (41%). The control treatment performed better in contained defects in terms of CAL and RBF. There was no significant difference in patient-reported outcomes between groups. CONCLUSIONS: At 2-year follow-up, the test and cotrol treatments were similarly effective in improving CAL, whereas the test treatment achieved a significantly greater RBF. In both treatments, favourable clinical, radiographic, and patient-reported outcomes can be sustained for at least 2 years. TRIAL REGISTRATION: The University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) 000025257.

Effects of CpG methylation on the thermal stability of c-kit2, c-kit*, and c-kit1 G-quadruplex structures.

In genomic DNA, G-quadruplex (G4)-forming DNA can form either a duplex or G4 structure, suggesting that understanding the factors regulating G4 formation is important for revealing the cellular functions controlled by G4 formation. Cytosine DNA methylation in the CpG islands is known to play an important role in transcriptional regulation. Additionally, CpG methylation increases the thermal stability of G4 structures such as BCL2 and VEGF G4. In this study, we evaluated the effects of CpG methylation in three G4 structures (c-kit2, c-kit*, and c-kit1) produced by the c-KIT promoter. Each was analyzed using circular dichroism (CD) melting analysis. The results demonstrate that CpG methylation does not alter the thermal stability of c-kit2 G4 structure when formed in the presence of K+; a single-CpG methylation at C1 or C11 decreases the thermal stability of any c-kit2 G4 structure formed in the presence of Na+ and Mg2+ while methylation at C5 increases the thermal stability; CpG methylation does not alter the thermal stability of c-kit1 or c-kit* G4 structures formed in the presence of K+; and the c-kit1 and c-kit* G4-forming oligonucleotides do not form G4 structures in the presence of Na+ and Mg2+. These results provide important clues for understanding the regulatory mechanisms underlying the formation of CpG methylation-induced G4 structures.

Amide-functionalised phosphonium-based ionic liquids as ligands for rhodium(iii) extraction.

Seven new amide-functionalised phosphonium-based ionic liquids (APILs) with chloride anions are synthesised and applied to extraction of rhodium(iii) from HCl solution. The effects of structural modification of the APILs on the extraction performance are examined by liquid-liquid extraction using toluene as a diluent and the results compared with those for trihexyltetradecylphosphonium chloride ([P66614][Cl]), a typical commercial extractant. The performance of the APILs as rhodium(iii) extractants is influenced by three main factors: (1) the length of the alkyl chains attached to the P atom; (2) the length of the linker between the amide and phosphonium moiety; and (3) the type of amide group. A novel ligand, [3°C2P444][Cl], had outstanding performance in the effective recovery of rhodium(iii). Extraction of rhodium(iii) from a 1.0 mol dm-3 HCl solution with 0.5 mol dm-3 [3°C2P444][Cl] proceeded quantitatively (>98%) and the extraction efficiency was higher than that of the commercial extractant [P66614][Cl]. The mechanism of rhodium(iii) extraction by [3°C2P444][Cl] was investigated by slope analysis, UV-vis, and FT-IR spectroscopy. These results indicate that [RhCl4(H2O)2]- in aqueous solution is extracted by [3°C2P444][Cl] through an anion-exchange mechanism and slowly converted into a dimer, [Rh2Cl9]3-, in the organic phase.

Treatment with functionalized designer self-assembling peptide hydrogels promotes healing of experimental periodontal defects.

BACKGROUND/OBJECTIVES: It has been reported that self-assembling peptide (SAP) hydrogels with functionalized motifs enhance proliferation and migration of host cells. How these designer SAP hydrogels perform in the treatment of periodontal defects remains unknown. This study aimed to test the potential of local application of designer SAP hydrogels with two different functionalized motifs in the treatment of experimental periodontal defects. MATERIAL AND METHODS: In vitro, viability/proliferation of rat periodontal ligament-derived cells (PDLCs) cultured on an SAP hydrogel RADA16 and RADA16 with functionalized motifs, PRG (integrin binding sequence) and PDS (laminin cell adhesion motif), was assessed. Cell morphology was analyzed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). In vivo, standardized periodontal defects were made mesially in the maxillary first molars of Wistar rats. Defects received RADA16, PRG, PDS or left unfilled. At 2 or 4 weeks postoperatively, healing was assessed by microcomputed tomography, histological and immunohistochemical methods. RESULTS: Viability/proliferation of PDLCs was significantly greater on PRG than on RADA16 or PDS at 72 hours. rPDLCs in the PRG group showed enhanced elongations and cell protrusions. In vivo, at 4 weeks, bone volume fractions in the PRG and PDS groups were significantly greater than the RADA16 group. Histologically, bone formation was more clearly observed in the PRG and PDS groups compared with the RADA16 group. At 4 weeks, epithelial downgrowth in the hydrogel groups was significantly reduced compared to the Unfilled group. In Azan-Mallory staining, PDL-like bundles ran in oblique direction in the hydrogel groups. At 2 weeks, in the area near the root, proliferating cell nuclear antigen (PCNA)-positive cells were detected significantly more in the PRG group than other groups. At 4 weeks, in the middle part of the defect, a significantly greater level of vascular endothelial growth factor (VEGF)-positive cells and α-smooth muscle actin (SMA)-positive blood vessels were observed in the PRG group than in other groups. CONCLUSION: The results indicate that local application of the functionalized designer SAP hydrogels, especially PRG, promotes periodontal healing by increasing cell proliferation and angiogenesis.

Treatment of Chronic Periodontitis with Recombinant Human Fibroblast Growth Factor-2 and Deproteinized Bovine Bone Mineral in Wide Intrabony Defects:12-month Follow-up Case Series.

Clinical use of 0.3% recombinant human fibroblast growth factor (rhFGF)-2 for periodontal regeneration received formal approval in Japan in 2016. The combination of growth factor and bone graft material is used to enhance periodontal healing in regenerative therapy. The exact effects of combination therapy on periodontal healing remain unknown, however. Here, we report three cases of chronic periodontitis treated with the combination of rhFGF-2 and deproteinized bovine bone mineral (DBBM). Following initial periodontal therapy, periodontal regenerative therapy using rhFGF-2 in combination with DBBM was performed to treat wide intrabony defects. Periodontal parameters and radiographic bone fill were reevaluated at 3 months, 6 months, and 1 year postoperatively. Oral health-related quality of life (OHRQL) was assessed as a patient-reported measure of outcome. At 1 year postoperatively, probing pocket depth and clinical attachment level showed a significant improvement in comparison with at baseline. An improvement was also noted in radiographic evidence of bone fill and total OHRQL scores. Combination therapy yielded clinically favorable results in the present cases.

Vibronic coupling density analysis and quantum dynamics simulation for singlet fission in pentacene and its halogenated derivatives.

We theoretically investigate microscopic origins of vibronic coupling (VC) contributing to singlet fission (SF) dynamics in pentacene and its halogenated derivatives. The features of VCs related to diabatic exciton states and interstate electronic couplings (Holstein and Peierls couplings, respectively) are interpreted by the VC density (VCD) analysis, which allows one to clarify the relationship between the chemical structure and VC as spatial contribution. It is found for the pentacene dimer face-to-edge configuration in a herringbone crystal that characteristic intermolecular vibrations with low frequencies exhibit strong Holstein couplings for the intermediate charge-transfer (CT) exciton states as well as Peierls couplings. From VCD analysis, the comprising density of the intermolecular CT and that of the intermolecular vibration are found to be constructively mixed in the intermolecular space, leading to the enhancement of VC. Moreover, in order to assess the chemical modification manner for controlling VC, we design several halogenated pentacene derivatives with slip-stack configurations. Our strategy to enhance VCD by halogenation is found to be rational, whereas the peaks of VC spectra for the CT states in the slip-stack packings are observed in high frequency regions. We compare their SF dynamics based on the quantum master equation explicitly including the exciton state-dependent VCs. From the analysis on relative relaxation factors between the adiabatic exciton states, their difference in the SF rate is highlighted by exciton configurations in addition to VCs. The present study is expected to be a first step toward efficient SF based on the design of VC in terms of both the chemical structure and intermolecular packing.

Quantum design for singlet-fission-induced nonlinear optical systems: Effects of π-conjugation length and molecular packing of butterfly-shaped acenes.

Theoretical molecular design of efficient nonlinear optical (NLO) systems using singlet fission (SF) is performed for butterfly-shaped acenes with/without nitrogen and sulfur substitutions using quantum chemical calculations, exciton dynamics simulations, and Marcus theory. It is found that these large systems meet the energy level matching conditions of efficient SF and exhibit superior third-order NLO properties (second hyperpolarizability γ at the molecular scale) to a typical SF molecule, pentacene. In addition, we investigate SF rates and γ in the correlated triplet pair [1(TT)] state generated by SF for various slip-stacked dimer models of these systems. For molecular packing with relatively large 1(TT) yields, a significant increase in γ/monomer in the 1(TT) state is observed, which is in good agreement with the electronic-coupling-based design guidelines obtained from our previous study. In particular, the butterfly-shaped acenes involving heteroatoms are found to exhibit a significant increase in γ/monomer as compared to the other systems. By analyzing the excitation properties in the 1(TT) state and intermolecular orbital interactions, we clarify the origin of such γ enhancement. The obtained results contribute to the construction of design guidelines for efficient SF-induced-NLO materials and demonstrate that butterfly-shaped acenes have the potential to surpass conventional NLO systems.

Theoretical Molecular Design of Phenanthrenes for Singlet Fission by Diazadibora-Substitution.

Based on the valence configuration interaction (VCI) model and quantum chemical calculations, we theoretically investigate the potential of diazadibora-substituted phenanthrenes [(BN)2-phenanthrenes] as novel singlet fission (SF) chromophores. (BN)2-substitution to phenanthrene is performed to exhibit a captodative effect, which is found to enhance both diradical character and exchange integral. These enhanced parameters induced by (BN)2-substitution are shown to bring energetically favorable SF with high triplet excitation energies. In order to reveal the relationship between diradical character and positions replaced by (BN)2, analyses based on the VCI model, odd-electron density, and resonance structures are conducted. Accordingly, a concrete design principle, which is inherent in and is understandable from the topology of (BN)2-phenanthrene, is presented. Furthermore, design strategies to fine-tuning of the diradical character are newly demonstrated based on the additional introduction of π-donor and π-acceptor. The present results provide feasible candidate molecules and novel design strategies toward the discovery of bright SF chromophores for the application to efficient organic solar cells.