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We developed a hybridization-based CpG methylation level detection system using methyl-CpG binding domain (MBD)-fused fluorescent protein, which is applicable to genome-wide DNA modification detection using microarrays.

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.

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 (

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.

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.

Non-Markov effects on singlet fission (SF) dynamics of model pentacene dimers are investigated using the second-order time-convolutionless quantum master equation method with state-independent (SI)...

We theoretically investigate significant impacts of diradical character and exchange integral on the first singlet (S1) and triplet (T1) excitation energies related to singlet fission, from which a...

Efficient and selective transport of rhodium(III) across a polymer inclusion membrane (PIM) from a 0.1 mol dm-3 HCl feed solution, also containing iron(III), to a receiving solution containing 0.1 mol dm-3 HCl and 4.9 mol dm-3 NH4Cl was achieved using a phosphonium-type ionic liquid, trioctyl(dodecyl)phosphonium chloride (P88812Cl), as the metal ion carrier. The optimum PIM composition for the Rh(III) transport was 50 wt % poly(vinylidene-co-hexafluoropropylene) (PVDF-HFP), 30 wt % P88812Cl, and 20 wt % plasticizer 2-nitrophenyl octyl ether (2NPOE). The driving force for the Rh(III) transport was suggested to be the concentration difference of the chloride ion between the feed and the receiving solutions. More than 70% rhodium(III) could be recovered from the receiving solution, and no transport of iron(III) was observed; however, the two metal ions cannot be separated by liquid-liquid extraction. This is the first report of selective transport of rhodium(III) across a polymer inclusion membrane.

In order to reduce the amount of CO2 emission during carburizing while keeping the advantages of case hardening, a surface cooled induction heating fast tempering process has been developed for the production of bearing elements. This process is applied to SUJ2 bearing steel, followed by conventional hardening. By surface cooled induction heating fast tempering, high temperature at the center of the test piece and relatively low temperature at the surface cause a difference in tempering temperature, leading to compressive residual stresses at the surface because of the difference in volume change during tempering. As a result, the bearing made by this process has the same rolling contact fatigue life as a carburized bearing due to high compressive residual stress. Process simulation of surface cooled induction heating fast tempering shows that residual stress is mainly caused by the difference in volume change of martensite during this process between the surface and the center. However, the decomposition of retained austenite appears to shift residual stress toward tensile stress.

N6-methyladenine (m6dA) has been recently discovered in eukaryotic genomic DNA. However, there have been few reports on its biological roles. G-quadruplex (G4) is a non-canonical nucleic acid structure formed by the stacking of G-tetrads. G4-forming sequences are enriched with cis-regulatory elements in genomic DNA and the G4 structures have important roles in various cellular functions. We previously reported that CpG methylation stabilized vascular endothelial growth factor (VEGF) G4 structure. Here we report that m6dA modification destabilizes the human c-kit1 G4 structure. These results suggest that epigenetic modifications may affect G4 formation in order to regulate the biological functions.

In this study, we theoretically investigate the aromatic and open-shell characteristics of carbon nanobelts (CNBs) composed of five- and six-membered rings. We have designed nanobelts composed of indeno[1,2-b]fluorene ([1,2-b]IF) units, which are referred to as [N]IF-CNB (N: the number of five-membered rings). The number of π-electrons, nπ, in neutral [N]IF-CNB is 7N, and thus depending on N and charge states, nπ can be 4n + 2 and 4n. Quantum chemical calculations on neutral [6]IF-CNB and [8]IF-CNB and dicationic [8]IF-CNB2+ have revealed that they are expected to exhibit unique aromatic and open-shell characteristics depending on nπ, there are several analogies of the electronic structures in [N]IF-CNB to those in [N]annulene. Delocalized and intermediate open-shell electronic structures of [N]IF-CNB are also useful to drastically change the third-order nonlinear optical properties. These results suggest that theoretically designed [N]IF-CNB can be attractive and challenging targets of organic synthesis for realizing novel open-shell functional conjugated macrocycles.

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.

Hydaticus pacificus conspersus Régimbart, 1899 (Coleoptera: Dytiscidae), is currently considered “endangered,” according to the national Red List of Japan. Herein, the developmental period of the immature stages of H. pacificus conspersus during laboratory rearing is described in detail for the first time. We found that this species deposits eggs not only on aquatic plants but also on the surface of driftwood. The observed durations of developmental stages were as follows: egg period – 3 days; first instar – 1–2 days (1.7 ± 0.5); second instar – 1–4 days (2.4 ± 0.6); third instar – 4–7 days (5.0 ± 0.7 days); and construction of a pupal chamber to emergence: 8–13 days (9.6 ± 0.8). Based on these observed results, H. pacificus conspersus has one of the shortest larval developmental periods among the Dytiscidae species. The short larval period and the ability to lay eggs on substrates other than plants may be an adaption of the species to increase reproductive success in unstable environments.

We report on the separation and recovery of scandium(III) from sulfate solutions using solvent extraction and a membrane transport system utilizing newly synthesized amic acid extractants. Scandium(III) was quantitatively extracted with 50 mmol dm–3N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]glycine (D2EHAG) or N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]phenylalanine (D2EHAF) in n-dodecane at pH 2 and easily stripped using a 0.5 mol dm–3 sulfuric acid solution. The extraction mechanisms of scandium(III) extraction with D2EHAG and D2EHAF were examined, and it was established that scandium(III) formed a 1:3 complex with both extractants (HR), that is, Sc(SO4)2–aq + 1.5(HR)2org ⇄ Sc(SO4)R(HR)2org + H+aq + SO42–aq. The equilibrium constants of extraction were evaluated to be 4.87 and 9.99 (mol dm–3)0.5 for D2EHAG and D2EHAF, respectively. D2EHAG and D2EHAF preferentially extracted scandium(III) with a high selectivity compared to common transition metal ions under high acidic conditions (0 < pH ≤ 3). In addition, scandium(III) was quantitatively transported from a feed solution into a 0.5 mol dm–3 sulfuric acid receiving solution through a polymer inclusion membrane (PIM) containing D2EHAF as a carrier. Scandium(III) was completely separated thermodynamically from nickel(II), aluminum(III), cobalt(II), manganese(II), chromium(III), calcium(II), and magnesium(II), and partially separated from iron(III) kinetically using a PIM containing D2EHAF as a carrier. The initial flux value for scandium(III) (J0,Sc = 1.9 × 10–7 mol m–2 s–1) was two times higher than that of iron(III) (J0,Fe = 9.3 × 10–8 mol m–2 s–1).

To facilitate ex-situ conservation of predaceous endangered insect species large numbers of live prey organisms are typically collected from the natural environment, potentially exacerbating biodiversity decline. To address this issue, two endangered beetle species (Cybister chinensis Motschulsky, 1854 and Cybister lewisianus Sharp, 1873) were reared using only laboratory-bred food. The beetle larvae were provided with laboratory-bred two-spotted cricket (Gryllus bimaculatus DeGeer; Orthoptera: Gryllidae) larvae as prey. The survival rates of the beetle larvae were as follows: C. chinensis, first instar larvae, 100% (n = 25), second instar larvae, 100% (n = 25), and third instar larvae, 92% (n = 23); C. lewisianus, first instar larvae 96% (n = 24), second instar larvae 92% (n = 23), and third instar larvae 92% (n = 23). The body length of adult insects fed laboratory-bred crickets did not significantly differ compared to the body length of field-collected adults.

Using quantum chemical calculations and exciton dynamics simulations, we investigate the static second hyperpolarizability γ [the third-order nonlinear optical (NLO) property at the molecular scale] of slip-stacked pentacene dimer models in the correlated-triplet-pair [1(TT)] state created from the singlet excited state in the singlet fission (SF) process. It is found that the SF induces significant (∼20 times at maximum) enhancement of γ/monomer in the 1(TT) state as compared to that in the singlet ground state. The origin of the remarkable enhancement of γ/monomer is revealed by analyzing the γ density distribution and the intermolecular orbital interaction. Furthermore, we clarify molecular packings suitable for highly efficient SF and largely enhanced γ values of a novel class of SF-induced NLO systems, which have promising potential to surpass the conventional NLO systems.

This paper reports on the selective separation and recovery of gold ions from leachates of discarded mobile phones using liquid-liquid extraction and a polymer inclusion membrane (PIM) transport system. The collected mobile phones were crushed by a mill and the obtained powder was calcinated. After leaching with aqua regia, the metal composition of the leachate was analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). The analysis results confirmed that the mobile phone waste contained 397 g/ton of gold. Liquid-liquid extraction and PIM-based separation procedures for the selective recovery of gold(III) from synthetic and actual leachates were developed. The extracting organic solution and the PIM incorporated the newly synthesized extractant N-[N,N-di(2-ethylhexyl)aminocarbonylmethyl]glycine (D2EHAG) which exhibited high selectivity for the gold(III) ion over the other metal ions present in much higher concentrations in the leachates. The compositions of the feed and receiving solutions in both the liquid-liquid and PIM based extraction and back-extraction of the gold(III) ions were optimized. It was established that optimal extraction required a HCl concentration in the feed solution of 2 mol/L and that a receiving solution containing 0.1 M thiourea in 1 M HCl was capable of back-extracting gold(III) quantitatively. Membrane transport experiments with a synthetic leachate as the feed solution demonstrated that 96% of the gold(III) ions was selectively transported into the receiving solution of the transport cell thus leaving all other metal ions in the leachate.