Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole.

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.

Development of HGF-binding aptamers with the combination of G4 promoter-derived aptamer selection and in silico maturation.

We describe the selection of aptamers based on bioinformatics-based approaches without Systematic Evolution of Ligands by EXponential enrichment (SELEX). SELEX is a potent method; however, it is time intensive and the PCR-amplification step, which is essential step for SELEX, leads to the loss of good aptamers. We have developed an aptamer-screening method, G4 promoter-derived aptamer selection (G4PAS), and an aptamer-improving method, in silico maturation (ISM). They are based on in silico sequence selection and computer assisted directed evolution, respectively. In this study, we succeeded in identifying new aptamers against hepatocyte growth factor (HGF) by G4PAS as well as improving the specificity of the HGF aptamers by ISM. Using ISM improved the specificity of the aptamer for HGF by up to 45-fold in comparison with the original aptamer. These methods enable easy and efficient identification of good aptamers, and the combination of G4PAS with ISM can thus serve as a potent approach for aptamer identification. Biotechnol. Bioeng. 2017;114: 2196-2203. © 2017 Wiley Periodicals, Inc.

Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA.

G-quadruplexes (G4s) are noncanonical DNA/RNA structures formed by guanine-rich sequences. Recently, G4s have been found not only in aptamers but also in the genomic DNA and transcribed RNA. In this study, we identified new RNA oligonucleotides working as aptamers by focusing on G4-forming RNAs located within the pre-mRNA. We showed that the G4 in the 5' UTR and first intron of VEGFA bound to the protein encoded in VEGFA gene, VEGF165, with high affinity. Moreover, G4-forming RNAs located within the PDGFA and the PDGFB introns bound to PDGF-AA and PDGF-BB, respectively, indicating that G4 in the pre-mRNA could be an aptamer. It had been reported that the putative G4-forming RNA sequences are located in some parts of most genes, thus our strategy for aptamer identification could be applicable to other proteins. It has been reported that some G4-forming RNAs in 5' UTRs are involved in translation control; however, G4-forming excised intronic RNA function has not been revealed previously. Therefore, these findings could not only contribute to the identification of RNA aptamers but also provide new insights into the biological functioning of G4-forming RNAs located within intronic RNA sequences.

Data on G-quadruplex topology, and binding ability of G-quadruplex forming sequences found in the promoter region of biomarker proteins and those relations to the presence of nuclear localization signal in the proteins.

Aptamer is a nucleic acid ligand which specifically binds to its target molecule. Previously, we have designed an identification method of aptamer called "G-quadruplex (G4) promoter-derived aptamer selection (G4PAS)" [1]. In G4PAS procedure, putative G4 forming sequences (PQS) were explored in a promoter region of a target protein in human gene through computational analysis, and evaluated binding ability towards the gene product encoded in the downstream of the promoter. We investigated the topology of the obtained PQSs by circular dichroism measurement, as well as their binding ability against its target protein by surface plasmon resonance measurement and gel-shift assay. Additionally, the presence of nuclear localization signal in the target protein was predicted in silico. This data set summarized all the PQS sequences, their biochemical characteristics, and the presence of nuclear localization signal to address the possibility of binding of these PQS region to the target proteins in vivo. Those data should contribute to increase the success rate of G4PAS. Moreover, considering the G4 motifs in genomic DNA are suggested to be involved in vivo gene regulation [2], [3], this data set is also potentially beneficial for the cell biology field.

Sonographic detection of physiological lower leg oedema in the late afternoon in healthy young women.

Lower leg oedema occurs physiologically in the evening after daytime activity. Various oedema-related sonographic findings have recently been reported, but this physiological oedema has not been evaluated quantitatively using imaging examinations. The present study investigated whether sonography could detect physiological lower leg oedema, comparing measured values between the morning and late afternoon. Diameters of leg veins were also measured as a possible source of leg oedema. Subjects comprised 55 healthy young women (mean age, 21 ± 1 years). Oedema-related findings such as papillary dermis thickness, subcutaneous adipose tissue thickness and echogenicity (as estimated in grey-scale using image analysis software) increased in the late afternoon when compared with those in the morning (1.4 [1.1-1.7] mm vs. 1.4 [1.1-1.8] mm, p < .01; 7.3 [6.0-8.1] mm vs. 7.3 [6.1-8.3] mm, p < .05; and 37.3 [31.5-39.4] vs. 39.8 [35.7-44.1], p < .01, respectively). Diameters of leg veins such as the great saphenous vein, small saphenous vein and dorsal vein of the foot were all reduced towards late afternoon (p < .01 each). Sonography quantitatively and precisely detected physical changes associated with physiological lower leg oedema after daytime activity in healthy young women.

Photosynthetic acclimation of terrestrial and submerged leaves in the amphibious plant Hygrophila difformis.

Hygrophila difformis, a heterophyllous amphibious plant, develops serrated or dissected leaves when grown in terrestrial or submerged conditions, respectively. In this study, we tested whether submerged leaves and ethylene-induced leaves of the heterophyllous, amphibious plant H. difformis have improved photosynthetic ability under submerged conditions. Also, we investigated how this amphibious plant photosynthesizes underwater and whether a HCO3 - transport system is present. We have analysed leaf morphology, measured underwater photosynthetic rates and HCO3 - affinity in H. difformis to determine if there are differences in acclimation ability dependent on growth conditions: terrestrial, submerged, terrestrial treated with ethylene and submerged treated with an ethylene inhibitor. Moreover, we measured time courses for changes in leaf anatomical characteristics and underwater photosynthesis in terrestrial leaves after submersion. Compared with the leaves of terrestrially grown plants, leaf thickness of submerged plants was significantly thinner. The stomatal density on the abaxial surface of submerged leaves was also reduced, and submerged plants had a significantly higher O2 evolution rate. When the leaves of terrestrially grown plants were treated with ethylene, their leaf morphology and underwater photosynthesis increased to levels comparable to those of submerged leaves. Underwater photosynthesis of terrestrial leaves was significantly higher by 5 days after submersion. In contrast, leaf morphology did not change after submergence. Submerged leaves and submerged terrestrial leaves were able to use bicarbonate but submerged terrestrial leaves had an intermediate ability to use HCO3 - that was between terrestrial leaves and submerged leaves. Ethoxyzolamide, an inhibitor of intracellular carbonic anhydrase, significantly inhibited underwater photosynthesis in submerged leaves. This amphibious plant acclimates to the submerged condition by changing leaf morphology and inducing a HCO3 - utilizing system, two processes that are regulated by ethylene.

Regulation of CCl4-induced liver cirrhosis by hepatically differentiated human dental pulp stem cells.

Liver transplantation is the most effective treatment for treating liver cirrhosis. However, a limited number of donors, graft rejection, and other complications can undermine transplant success. It is considered that cell transplantation is an alternative approach of liver transplantation. We previously developed a protocol for hepatic differentiation of cluster of differentiation 117+ stem cells isolated from human exfoliated deciduous tooth pulp (SHEDs) under hydrogen sulfide exposure. These cells showed excellent hepatic function. Here, we investigated whether hepatocyte-like cell transplantation is effective for treating carbon tetrachloride (CCl4)-induced liver cirrhosis. SHEDs were hepatically differentiated, which was confirmed via immunological analyses and albumin concentration determination in the medium. Rats were intraperitoneally injected with CCl4 for and the differentiated cells were injected into rat spleen. Histopathological and immunohistochemical analyses were performed. Liver functions were serologically and pathologically determined. Quantitative real-time-polymerase chain reaction was implemented to clarify the treatment procedure of liver cirrhosis. In vitro-differentiated hepatocyte-like cells were positive for all examined hepatic markers. SHED-derived hepatocyte transplantation eliminated liver fibrosis and restored liver structure in rats. Liver immunohistochemical analyses showed the presence of human-specific hepatic markers, i.e., a large amount of human hepatic cells were very active in the liver and spleen. Serological tests revealed significant liver function recovery in the transplantation group. Expression of genes promoting fibrosis increased after cirrhosis induction but was suppressed after transplantation. Our results suggest that xenotransplantation of hepatocyte-like cells of human origin can treat cirrhosis. Moreover, cell-based therapy of chronic liver conditions may be an effective option.

Regeneration of insulin-producing islets from dental pulp stem cells using a 3D culture system.

AIM: In this study, we aimed to establish the differentiation protocol of dental pulp stem cells (DPSCs) into pancreatic islets using a 3D structure. MATERIALS & METHODS: DPSCs were differentiated in a 3D culture system using a stepwise protocol. Expression of ß-cell markers, glucose-stimulated insulin secretion, and PI3K/AKT and WNT pathways were compared between monolayer-cultured pancreatic cells and islets. RESULTS: Islet formation increased insulin and C-peptide production, and enhanced the expression of pancreatic markers. Glucose-dependent secretion of insulin was increased by islets. Pancreatic endocrine markers, transcriptional factors, and the PI3K/AKT and WNT pathways were also upregulated. CONCLUSION: Pancreatic islets were generated from DPSCs in a 3D culture system. This system could provide novel strategies for controlling diabetes through regenerative medicine.

Effect of Gum Chewing Frequency on Oxygenation of the Prefrontal Cortex.

Since increased cerebral oxygenation reflects cerebral activation, this study investigated the effect of mastication frequency on prefrontal cortex oxygenation. Eleven young volunteers (nine women, two men; M age = 20.9 years, SD = 0.9) carried out three trials in which they were asked to chew a tasteless gum for 3 min at varying (rates of mastication frequency: 30, 70, and 110). Breaks of 2 min each were interleaved between trials. The oxygenation of the left prefrontal cortex was monitored by near-infrared spectroscopy. We found a significant increase in cortical oxygenation during gum chewing in all three conditions ( p < .05), compared with a resting level; we also found a significant difference between the Fast and Slow chewing conditions, and between the Fast and Normal (70 rpm) conditions, both findings seemingly related to activation of a motor command in frontal brain regions. To our knowledge, this is the first report on the effect of mastication frequency on cerebral oxygenation. Possible implications of this finding are discussed.

Aptamer selection based on G4-forming promoter region.

We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as "G4 promoter-derived aptamer selection (G4PAS)." Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10(-7) M, 6.3 × 10(-9) M, and 4.4 × 10(-7) M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.

Nonexploratory movement and behavioral alterations in a thalidomide or valproic acid-induced autism model rat.

Autism is a behaviorally characterized disorder with impairments in social interactions, as well as stereotyped, repetitive patterns of behaviors and interests. Exposure of rat fetuses to thalidomide (THAL) or valproic acid (VPA) on the ninth day of gestation has been reported as a useful model for human autism. We have shown that early serotonergic neural development is disrupted in these rats. In the current study, we used a radial maze and open field experimental paradigm to investigate whether these rats present behavioral and/or learning aberrations. THAL (500mg/kg), VPA (800mg/kg), or vehicle was administered orally to E9 pregnant rats at 7-10 weeks of age. Although the mean number of correct and incorrect arm choices in the initial eight arm choices did not differ between control and teratogen-exposed groups, achievement of learning (seven or eight consecutive correct choices for 3 consecutive days for individual rats) seemed to be impaired in teratogen-exposed groups. Interestingly, average time to explore the maze task was shorter in the teratogen-exposed groups, indicating that correct choice might be due to mere coincidence (i.e., nonexploratory movement). Unexpectedly, no significant differences were observed in social interaction in these rats. These results indicate that prenatal exposure to THAL and VPA might alter behavior in a manner that is, in part, consistent with human autism.