Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs

Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.     

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.     

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor

Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 10³ cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.     

Baseline Assessment of Mesophotic Reefs of the Vitória-Trindade Seamount Chain Based on Water Quality,Microbial Diversity,Benthic Cover and Fish Biomass Data

Seamounts are considered important sources of biodiversity and minerals. However, their biodiversity and health status are not well understood; therefore, potential conservation problems are unknown. The mesophotic reefs of the Vitória-Trindade Seamount Chain (VTC) were investigated via benthic community and fish surveys, metagenomic and water chemistry analyses, and water microbial abundance estimations. The VTC is a mosaic of reef systems and includes fleshy algae dominated rhodolith beds, crustose coralline algae (CCA) reefs, and turf algae dominated rocky reefs of varying health levels. Macro-carnivores and larger fish presented higher biomass at the CCA reefs (4.4 kg per frame) than in the rhodolith beds and rocky reefs (0.0 to 0.1 kg per frame). A larger number of metagenomic sequences identified as primary producers (e.g., Chlorophyta and Streptophyta) were found at the CCA reefs. However, the rocky reefs contained more diseased corals (>90%) than the CCA reefs (~40%) and rhodolith beds (~10%). Metagenomic analyses indicated a heterotrophic and fast-growing microbiome in rocky reef corals that may possibly lead to unhealthy conditions possibly enhanced by environmental features (e.g. light stress and high loads of labile dissolved organic carbon). VTC mounts represent important hotspots of biodiversity that deserve further conservation actions.     

TAE226, a Bis-Anilino Pyrimidine Compound,Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells

TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.     

Exploiting the Extraordinary Genetic Polymorphism of Ciona for Developmental Genetics with Whole Genome Sequencing

Studies in tunicates such as Ciona have revealed new insights into the evolutionary origins of chordate development. Ciona populations are characterized by high levels of natural genetic variation, between 1 and 5%. This variation has provided abundant material for forward genetic studies. In the current study, we make use of deep sequencing and homozygosity mapping to map spontaneous mutations in outbred populations. With this method we have mapped two spontaneous developmental mutants. In Ciona intestinalis we mapped a short-tail mutation with strong phenotypic similarity to a previously identified mutant in the related species Ciona savignyi. Our bioinformatic approach mapped the mutation to a narrow interval containing a single mutated gene, α-laminin3,4,5, which is the gene previously implicated in C. savignyi. In addition, we mapped a novel genetic mutation disrupting neural tube closure in C. savignyi to a T-type Ca²⁺ channel gene. The high efficiency and unprecedented mapping resolution of our study is a powerful advantage for developmental genetics in Ciona, and may find application in other outbred species.     

N′-[4-(dipropylamino)benzylidene]-2-hydroxybenzohydrazide is a dynamin GTPase inhibitor that suppresses cancer cell migration and invasion by inhibiting actin polymerization

Dynasore, a specific dynamin GTPase inhibitor, suppresses lamellipodia formation and cancer cell invasion by destabilizing actin filaments. In search for novel dynamin inhibitors that suppress actin dynamics more efficiently, dynasore analogues were screened. N′-[4-(dipropylamino)benzylidene]-2-hydroxybenzohydrazide (DBHA) markedly reduced in vitro actin polymerization, and dose-dependently inhibited phosphatidylserine-stimulated dynamin GTPase activity. DBHA significantly suppressed both the recruitment of dynamin 2 to the leading edge in U2OS cells and ruffle formation in H1299 cells. Furthermore, DBHA suppressed both the migration and invasion of H1299 cells by approximately 70%. Furthermore, intratumoral DBHA delivery significantly repressed tumor growth. DBHA was much less cytotoxic than dynasore. These results strongly suggest that DBHA inhibits dynamin-dependent actin polymerization by altering the interactions between dynamin and lipid membranes. DBHA and its derivative may be potential candidates for potent anti-cancer drugs.     

Secretion of inflammatory factors from chondrocytes by layilin signaling

Layilin (LAYN) is thought to be involved in reorganization of cytoskeleton structures, interacting with merlin, radixin, and talin. Also, LAYN is known to be one of the receptors for hyaluronic acid (HA).     

The role of crown architecture, leaf phenology and photosynthetic activity in promoting complementary use of light among coexisting species in temperate forests

Mixed forests comprising multiple tree species with contrasting crown architectures, leaf phenologies, and photosynthetic activity, tend to have high ecosystem productivity. We propose that in such forests, differentiation among coexisting species in their spatial and temporal strategies for light interception, results in complementary use of light. Spatial differentiation among coexisting tree species occurs as a result of adaptation of crown architecture and shoot/leaf morphology to the spatially variable light conditions of the canopy, sub-canopy, and understory. Temporal differentiation occurs as a result of variation in leaf phenology and photosynthetic activity. The arrangement of leaves in both space and time is an important aspect of plant strategies for light interception and determines photosynthetic carbon gain of the plant canopy. For example, at the shoot level, morphological and phenological differentiation between long and short shoots reflects their respective shoot functions, indicating that spatial and temporal strategies for light interception are linked. Complementary use of light is a consequence of the spatiotemporal differentiation in light interception among coexisting species. Because coexisting species may show differentiation in strategies for resource acquisition (functional diversification) or convergence with respect to some limiting resource (functional convergence), the relative importance of various crown functions and their contribution to growth and survival of individuals need to be evaluated quantitatively and compared among coexisting species.     

How did the exposed seafloor function in postglacial northward range expansion of <Emphasis Type="Italic">Kalopanax septemlobus</Emphasis>? Evidence from ecological niche modelling

We attempted to clarify how coastal lands temporarily exposed during the last glacial maximum (LGM) contributed to the northward colonisation of Kalopanax septemlobus (Thunb. ex Murray) Koidzumi during the postglacial period in the Japanese Archipelago. Distribution records in 30-arc-s pixels were related to bioclimate variables using the maximum entropy technique to model the ecological niche of this species. Bioclimatic conditions in the exposed coastal lands during the LGM were reconstructed based on simulated palaeoclimate and fine-resolution marine topography. Potential distribution ranges were then estimated under the climatic conditions during the LGM, mid-Holocene and the present. The ecological niche of this species was influenced mainly by the temperature component of the bioclimates, leading to northward range shift after the LGM as the climate warmed. On average, 26% of the potential range of K. septemlobus during the LGM was located on the exposed seafloor. The northern edges of the species range on both sides of Honshu Island were estimated by up to several hundred kilometres farther north, when compared to the prediction assuming no sea level change. Application of ecological niche modelling provided new insight into the role of exposed seafloor as cryptic glacial refugia for this species, which has never been evidenced by fossil records. In a temperate island system characterised by long coastlines, the northern exposed seafloor would have been more important in terms of harbouring the putative source populations for northward directional colonisation during the postglacial period.