Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

Assessment of Demineralization Inhibition Effects of Dentin Desensitizers Using Swept-Source Optical Coherence Tomography.

The purpose of this study was to evaluate the mechanism of action and the inhibiting effects of two types of desensitizers against dentin demineralization using pre-demineralized hypersensitivity tooth model in vitro. In this study, we confirmed that a hypersensitivity tooth model from our preliminary experiment could be prepared by immersing dentin discs in an acetic acid-based solution with pH 5.0 for three days. Dentin discs with three days of demineralization were prepared and applied by one of the desensitizers containing calcium fluoro-alumino-silicate glass (Nanoseal, NS) or fluoro-zinc-silicate glass (Caredyne Shield, CS), followed by an additional three days of demineralization. Dentin discs for three days of demineralization (de3) and six days of demineralization (de6) without the desensitizers were also prepared. The dentin discs after the experimental protocol were scanned using swept-source optical coherence tomography (SS-OCT) to image the cross-sectional (2D) view of the samples and evaluate the SS-OCT signal. The signal intensity profiles of SS-OCT from the region of interest of 300, 500, and 700 µm in depth were obtained to calculate the integrated signal intensity and signal attenuation coefficient. The morphological differences and remaining chemical elements of the dentin discs were also analyzed using scanning electron microscopy and energy-dispersive X-ray spectroscopy. SS-OCT images of CS and NS groups showed no obvious differences between the groups. However, SS-OCT signal profiles for both the CS and NS groups showed smaller attenuation coefficients and larger integrated signal intensities than those of the de6 group. Reactional deposits of the desensitizers even after the additional three days of demineralization were observed on the dentin surface in NS group, whereas remnants containing Zn were detected within the dentinal tubules in CS group. Consequently, both CS and NS groups showed inhibition effects against the additional three days of demineralization in this study. Our findings demonstrate that SS-OCT signal analysis can be used to monitor the dentin demineralization and inhibition effects of desensitizers against dentin demineralization in vitro.

Construction and characterization of the PGN_0296 mutant of Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis produces gingipains (Kgp, RgpA, and RgpB), cysteine proteases involved in the organism's virulence, and pigmentation. We previously showed that deletion of the PGN_0297 and PGN_0300 genes reduced the proteolytic activity of gingipains. The role of the PGN_0296 gene, consisting of an operon with the PGN_0297 and PGN_0300 genes, is unclear. Herein, we examined the effect of PGN_0296 gene deletion on the proteolytic activity. Although the proteolytic activity of the gingipains did not decrease in the culture supernatant of a PGN_0296 gene deletion mutant (ΔPGN_0296), the growth was delayed.

Influence of 10-MDP concentration on the adhesion and physical properties of self-adhesive resin cements.

OBJECTIVES: Self-adhesive resin cements contain functional monomers that enable them to adhere to the tooth structure without a separate adhesive or etchant. One of the most stable functional monomers used for chemical bonding to calcium in hydroxyapatite is 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP). The aim of this study was to evaluate the influence of the10-MDP concentration on the bond strength and physical properties of self-adhesive resin cements. MATERIALS AND METHODS: We used experimental resin cements containing 3 different concentrations of 10-MDP: 3.3 wt% (RC1), 6.6 wt% (RC2), or 9.9 wt% (RC3). The micro-tensile bond strength of each resin cement to dentin and a hybrid resin block (Estenia C&B, Kuraray Noritake Dental) was measured, and the fractured surface morphology was analyzed. Further, the flexural strength of the resin cements was measured using the three-point bending test. The water sorption and solubility of the cements following 30 days of immersion in water were measured. RESULTS: The bond strength of RC2 was significantly higher than that of RC1. There was no significant difference between the bond strength of RC2 and that of RC3. The water sorption of RC3 was higher than that of any other cement. There were no significant differences in the three-point bending strength or water solubility among all three types of cements. CONCLUSIONS: Within the limitations of this study, it is suggested that 6.6 wt% 10-MDP showed superior properties than 3.3 wt% or 9.9 wt% 10-MDP in self-adhesive resin cement.

Regional hypoplasia of somatosensory cortex in growth-retarded mice (grt/grt).

Growth-retarded mouse (grt/grt) is a spontaneous mutant that is known as an animal model for primary congenital hypothyroidism caused by resistance to TSH signaling. The regional pattern of cerebral cortical hypoplasia was characterized in grt/grt mice. Ex vivo computed tomography (CT)-based volumetry was examined in four regions of the cerebral cortex, i.e., prefrontal, frontal, parietal and occipito-temporal regions, which were demarcated by structural landmarks on coronal CT images. A region-specific reduced volume of the parietal cortical region covering most of the somatosensory cortex was noted in grt/grt mice rather than in both heterozygous (grt/+) and wild-type (+/+) mice. We concluded that the cortical hypoplasia in grt/grt was seen in identical cortical regions corresponding to human congenital hypothyroidism.

D-glucosamine promotes transfection efficiency during electroporation.

D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes) in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP) in the cultured cells (osteoblasts; NOS-1 cells). The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.

Familial clustering of mice consistent to known pedigrees enabled by the genome profiling (GP) method.

Familial clustering without any prerequisite knowledge becomes often necessary in Behavioral Science, and forensic studies in case of great disasters like Tsunami and earthquake requiring body-identification without any usable information. However, there has been no well-established method for this purpose although conventional ones such as short tandem repeats (STR) and single nucleotide polymorphism (SNP), which might be applied with toil and moil to some extent. In this situation, we could find that the universal genome distance-measuring method genome profiling (GP), which is made up of three elemental techniques; random PCR, micro-temperature gradient gel electrophoresis (µTGGE), and computer processing for normalization, can do this purpose with ease when applied to mouse families. We also confirmed that the sequencing approach based on the ccgf (commonly conserved genetic fragment appearing in the genome profile) was not completely discriminative in this case. This is the first demonstration that the familial clustering can be attained without a priori sequence information to the level of discriminating strains and sibling relationships. This method can complement the conventional approaches in preliminary familial clustering.

Surface roughness and gloss of indirect composites etched with acidulated phosphate fluoride solution.

OBJECTIVE: To evaluate the surface characteristics of indirect composites etched with acidulated phosphate fluoride (APF) solution. MATERIAL AND METHODS. APF solution was applied to specimens of six composite materials. The surface morphological change was measured with a confocal laser scanning microscope. Gloss measurement and scanning electron microscope (SEM) observation were also performed. RESULTS: The APF-treated surface showed various patterns due to the difference in the filler incorporated in each composite. The surface properties could be evaluated precisely from the surface roughness parameters. The arithmetical mean deviation of roughness profile (Ra) parameter of the specimens treated with APF solution was higher than those of the specimens untreated with APF, except New Meta Color Infis (IF). The difference in etching influence of each composite material was shown conclusively in the maximum height of the profile (Rz) parameter. Gradia (GR) and Gradia Forte (GF) were etched three times more deeply than that of IF categorized microfilled composite. The mean width of the profile element (RSm) decreased significantly after APF treatment, except in IF. Gloss was reduced apparently in all materials, indicating that gloss reduction was sensitive to slight surface changes. CONCLUSIONS: Specific filler particles of prosthodontic composite materials were etched by APF application. The surface roughness parameters, such as Rz and RSm, properly described various surface topographic features. Gloss was strongly correlated to surface roughness, as defined by these parameters, and especially to the initial change of surface roughness.

Bond strength to bovine dentin of a composite core build-up material combined with four different bonding agents.

Clearfil DC Bond (DC) is a new single-step, dual-cure bonding agent. In this study, the shear bond strengths of a core build-up composite to dentin used with four bonding systems [DC, Unifil Core Self-Etching Bond (UC), Clearfil SE Bond (SE) and Cleafil tri-S Bond (TS)] were measured. The bonding ability after 7 days of storage and in vitro durability following 20,000 thermocycles were also evaluated. The bond strength of DC did not differ significantly from those of other bonding systems after 24 hours of storage. Another dual-cure bonding system, UC, showed a significant reduction of bond strength after 7 days of storage. On the other hand, the bond strength of TS, a light-cured bonding system with a similar composition to DC, was reduced significantly following 20,000 thermocycles. SE, a two-step light-cure bonding system in the same series as DC, provided superior bond strength under all conditions. Although DC showed a slightly lower bond strength than SE, there was no significant difference between DC and SE under all conditions. Consequently, DC may be a useful and effective bonding system for multiple composite resin restorations.

Chitosan monomer accelerates alkaline phosphatase activity on human osteoblastic cells under hypofunctional conditions.

Chitosan is a natural polyaminosaccharide that is extensively applied as an antitumor and antirheumatic drug. However, there are few reports about its effects on hypofunctional osteoblasts in vitro. We investigated the biological characteristics of a human osteoblastic cell line (NOS-1 cells) that was cultured with a chitosan monomer-containing medium under simulated microgravity conditions. After 7 days of cell incubation under the conventional conditions, the flasks were transferred to a microgravity simulator for 3 days. In the 0.005% chitosan monomer supplemented group, the marker enzyme of biological mineralization, the alkaline phosphatase (ALP) activity, was significantly higher compared with the control group (p<0.05). A cDNA microarray was performed to investigate the effects on the mRNA level by chitosan monomer, and the fluorescent signal was analyzed. The interferon gamma (IFN-gamma) receptor gene was detected with a signal ration of 2.2. The slight increase of IFN-gamma receptor expression was confirmed after 3 days of incubation according to RT-PCR analysis. Western blot analysis also showed the increased expression of IFN-gamma receptor. These results suggest that a supra-low concentration of chitosan monomer may increase the ALP activity of osteoblastic cells through the IFN-gamma receptor at the early phase of cell culture and recover the activity for biological mineralization under the hypofunctional condition.

Early gene expression analyzed by cDNA microarray and real-time PCR in osteoblasts cultured with chitosan monomer.

Chitosan has a variety of biological activities. Although it has been reported that chitosan promotes osteogenesis in bone lesions, little is known about how it modulates the hard tissue forming cells at the gene level. This study focused on gene expressions in osteoblasts cultured with a super-low concentration of chitosan monomer. cDNA probes were synthesized from isolated RNA and labeled with fluorescent dye. They were hybridized with Human 3.8 II cDNA microarray, and the fluorescent signal was analyzed. cDNA microarray analysis revealed that 10 genes concerning to various signaling-related molecules were expressed at > or =2.0-fold higher signal ratio levels in the experimental group when compared with the control group after 3 days. Real-time PCR analysis showed that chitosan monomer induced an increase in the expression of four signal transduction genes, mitogen-activated protein kinase (MAPK)K3, MAPKKK11, Rac1 and Shc1, together with the alkaline phosphatase gene. These results suggest that a super-low concentration of chitosan monomer could modulate the activity of osteoblastic cells through mRNA levels and that chitosan monomer directly affects signal transduction inside cells.

Chewing chitosan-containing gum effectively inhibits the growth of cariogenic bacteria.

OBJECTIVE: We have already reported that chitosan inhibited the growth of cariogenic bacteria in vitro. This study was designed to evaluate whether chewing gum, containing chitosan, can effectively suppressed the growth of oral bacteria (total bacteria, total Streptococci, mutans streptococci (MS)) in saliva. METHODS: Fifty healthy subjects, ranging in age from 19 to 32 years, were recruited from among the staff and students of Nagasaki University School of Dentistry. For the slab of gum study, the subjects chewed gum for 5 min and then rested for 5 min. Each subject chewed a total of eight pieces of gum, which was either supplemented with or without chitosan, for a total of 80 min. Two different types of gums were examined with at least 1 week as a rest period in between treatments. This in vivo study was carried out by the double blind comparison test. RESULTS: The amount of oral bacteria was found to significantly decrease in the chitosan group. Especially, the number of MS were maintained at about a 20% level in comparison to that before gum chewing, even at 1h after gum chewing. CONCLUSION: These findings suggest that a supplementation of chitosan to gum is an effective method for controlling the number of cariogenic bacteria in situations where it is difficult to brush one's teeth, such as when an individual is away from home all day or participating in outdoor training.

Synthesis and redox properties of trinuclear ruthenium-acetylide complexes with tri(ethynylphenyl)amine bridge.

Novel trinuclear ruthenium complexes have been prepared by using tri(4-ethynylphenyl)amine as a bridging ligand. Cyclic voltammetry of the trinuclear ruthenium complexes revealed stepwise quasi-reversible redox behavior of three ruthenium-acetylide species and the central triphenylamine unit, whereas the mononuclear analog showed two sequential quasi-reversible redox waves. The spectroelectrochemical UV-VIS spectral studies suggested that the 1e- oxidized triruthenium species was stable and showed a characteristic absorption at lambda(max) = 505 nm. Chemical oxidation of the triruthenium complex with ferrocenium hexafluorophosphate led to the isolation of the 1e- oxidized complex, the near-IR spectrum of which revealed an intervalence charge transfer band due to the electronic coupling among three ruthenium species. The 1e(-) oxidized triruthenium complexes can be classified as class II mixed-valence compounds.

Organocatalytic direct asymmetric aldol reactions in water.

We have developed direct asymmetric cross-aldol reactions that can be performed in water without addition of organic solvents. A bifunctional catalyst with a long hydrophobic alkyl chain efficiently catalyzed the reactions and afforded the desired aldol products in excellent yield with high enantiomeric excess, even when only an equal molar ratio of the donor and acceptor was used. These results reveal an effective design strategy for the development of aqueous organocatalytic systems.

Chitosan monomer promotes tissue regeneration on dental pulp wounds.

The present study was undertaken to evaluate the applicability of chitosan monomer (D-glucosamine hydrochloride) as a pulp capping medicament. Both in vitro and in vivo experiments were carried out to study the cell metabolism and wound healing mechanisms following the application of chitomonosaccharide. After 3 days of osteoblast culture, alkaline phosphatase (ALP) activity significantly increased in the chitosan group. Reverse transcription polymerase chain reaction analysis revealed that chitosan induced an increase in the expression of ALP mRNA after 3 days and bone morphogenetic protein-2 mRNA after 7 days of osteoblast incubation. Inflammatory cytokine, interleukin (IL)-8, synthesis in fibroblasts was strongly suppressed in the medium supplemented with chitosan monomer. Histopathological effects were evaluated in rat experiments. After 1 day, inflammatory cell infiltrations were observed to be weak when compared with the application of chitosan polymer. After 3 days, a remarkable proliferation of fibroblasts was seen near the applied chitosan monomer. The inflammatory cell infiltration had almost completely disappeared. After 5 days, the fibroblastic proliferation progressed, and some odontoblastic cells appeared at the periphery of the proliferated fibroblasts. These findings indicate that the present study is the first report that chitosan monomer acts as a biocompatibility stable medicament even at the initial stage of wound healing in comparison with the application of chitosan polymer.

Early gene expression analyzed by cDNA microarray and RT-PCR in osteoblasts cultured with water-soluble and low molecular chitooligosaccharide.

Chitosan has a variety of biological activities. However, little is known about how chitosan modulates the hard tissue forming cells. When we cultured an osteoblastic cell line in alpha-MEM supplemented with 10% FBS and 0.005% chitooligosaccharide for 3 days, alkaline phosphatase (ALP) activity was significantly high compared with the control culture group (p<0.05). This study was focused on gene expression in osteoblasts cultured with water-soluble chitooligosaccharide. cDNA probes were synthesized from isolated RNA and labeled with fluorescent dye. They were hybridized with Human 1.0((R)) cDNA microarray, and fluorescent signal was analyzed. cDNA microarray analysis revealed that 16 genes were expressed at >/=1.5-fold higher signal ratio levels in the experimental group compared with the control group after 3 days. RT-PCR analysis showed that chitosan oligomer induced an increase in the expression of two genes, CD56 antigen and tissue-type plasminogen activator. Furthermore, the expression of mRNAs for BMP-2 was almost identical in the experimental and control groups after 3 days of culture, but slightly increased after 7 days of culture with chitosan oligomer. These results suggest that a super-low concentration of chitooligosaccharide could modulate the activity of osteoblastic cells through mRNA levels and that the genes concerning cell proliferation and differentiation can be controlled by water-soluble chitosan.

Mineralization of matrix vesicles isolated from a human osteosarcoma cell line in culture with water-soluble chitosan-containing medium.

Chitosan is a natural bioactive material. Although it has been reported that chitosan promotes osteogenesis in bone lesions, little is known about how chitosan modulates this process. The present study was designed to investigate the effect of water-soluble chitosan relative to initiation of biologic mineralization, especially in the matrix-vesicles-(MVs) mediated process in vitro. A human osteoblastic cell line (NOS-1) was used. After 3 days of incubation, the number of cells and alkaline phosphatase (ALP) activity increased significantly in the chitosan group. RT-PCR analysis revealed that chitosan induced an increase in the expression of bone morphogenetic protein-2 mRNA after 7 days of incubation. MVs were isolated from NOS-1 cells using a collagenase digestion and ultracentrifugation method. ALP activity of MVs isolated from chitosan-supplemented cells was significantly higher than that of the control group. Furthermore, isolated MVs were incubated in medium supplemented with Na-beta-glycerophosphate without fetal bovine serum. Needle-like crystals were observed in association with MVs after 24 h of incubation. These needle-like crystals were densely accumulated in the chitosan group. The present findings suggest that water-soluble chitosan would promote osteoblast proliferation and differentiation and may be useful for the acceleration of initial biologic mineralization.

Spatiotemporal regulation of moesin phosphorylation and rear release by Rho and serine/threonine phosphatase during neutrophil migration.

Neutrophil motility is crucial to effective host defenses against microorganisms. While uropod retraction is a critical step in the migration of neutrophils, the underlying molecular mechanism is not well understood. Here, we show that inhibition of the Rho small GTPase with C3 exoenzyme prevented the retraction of trailing uropods, indicating that the process of rear release is mediated by a Rho signaling pathway. C3 exoenzyme caused marked elongation of directionally migrating neutrophils, suggesting an additional role for Rho in the maintenance of functional polarized cell shape. We also show that phosphorylation and dephosphorylation of the plasma membrane-actin filament cross-linker moesin are spatiotemporally controlled in migrating neutrophils. In particular, phosphorylation of moesin at threonine 558 depended on Rho activity. Videomicroscopy showed that dephosphorylation of this carboxy-terminal threonine preceded uropod retraction. Calyculin A, an inhibitor of type 1 and type 2A serine/threonine phosphatases, suppressed the moesin dephosphorylation and impaired uropod retraction in a dose-dependent manner. Cypermethrin, an inhibitor of type 2B serine/threonine phosphatase, had no such effects. The finding that Rho small GTPase and type 1/type 2A phosphatases are involved in rear release yields novel insights into the biochemical mechanisms of neutrophil migration.