Evaluation of photodynamic activity of C60/2-hydroxypropyl-β-cyclodextrin nanoparticles

The objective of this study is to evaluate the ability of C(60)/2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) naonparticles to generate reactive oxygen species (ROS) and to induce cell toxicity by the photoirradiation. C(60) nanoparticles were prepared by cogrinding with HP-β-CyD for 3 h at 4°C under reduced pressure. The photodynamic activity of C(60)/HP-β-CyD nanoparticles was evaluated by spectroscopic methods, including the electron spin resonance spin-trapping method, and by the cell viability test using Hela cells. C(60)/HP-β-CyD nanoparticles efficiently generated not only superoxide anion radical (O(2)(·-)) and hydroxyl radical (·OH), but also singlet oxygen ((1)O(2)) through photoirradiation. The ROS generation was enhanced by decreasing the mean particle diameter of C(60) nanoparticles, and the particle size smaller than 90 nm showed a high generation of ·OH and (1)O(2). In addition, HP-β-CyD enhanced the generation of (1)O(2), compared with polyvinylpyrrolidone (an effective solubillizer for C(60)), due to partial disposition of C(60) in the hydrophobic CyD cavity. Furthermore, C(60) /HP-β-CyD nanoparticles showed cell toxicity after the light irradiation, but no toxicity was observed without the light irradiation. Therefore, HP-β-CyD is useful for the preparation of stable C(60) nanoparticles with high ROS generation ability, and C(60)/HP-β-CyD nanoparticles are a promising photosensitizer for photodynamic therapy.     

N-Isopropyl-p-iodoamphetamine hydrochloride Is predominantly metabolized by CYP2C19

[(123)I]N-Isopropyl-p-iodoamphetamine hydrochloride ([(123)I]IMP) is clinically used to evaluate blood flow in the brain on single photon emission-computed tomography. This is a rare radiopharmaceutical that undergoes metabolism. The first step is reported to be [(123)I]p-iodoamphetamine formation. The drug-metabolizing enzyme(s) involved remain(s) unclear. This study examined the roles of human cytochrome P450 (P450) in the metabolism of nonradiolabeled IMP with the use of human liver microsomes (HLM) and recombinant human CYP1A1, -1A2, -1B1, -2A6, -2B6, -2C8, -2C9, -2C19, -2D6, -2E1, -3A4, and -3A5. Disappearance of IMP was examined because p-iodoamphetamine was not available. IMP (0.5 μM) time-dependently disappeared when HLM and NADPH-generating system were added to the reaction mixture. (S)-Mephenytoin (1 mM) inhibited the IMP disappearance by approximately 90%. The disappearance of IMP was predominantly catalyzed by recombinant CYP2C19, with K(m) and V(max) of 8.6 μM and 9.7 nmol · min(-1) · nmol P450(-1), respectively. IMP disappearance in CYP2C19-deficient HLM (CYP2C19*2/*2) was approximately 30% of that in the presence of HLM harboring wild-type CYP2C19, indicating that IMP is polymorphically metabolized by CYP2C19. High-performance liquid chromatography of the incubation mixture of IMP and CYP2C19 revealed an unidentified peak. As the area of the IMP peak decreased, the area of this unidentified peak increased in a time-dependent fashion. The peak was also detectable on incubation of IMP with HLM. Mass spectrometry revealed that the molecular weight of a compound in this unidentified peak was the same as that of p-iodoamphetamine. Thus, we demonstrated that IMP was predominantly metabolized by CYP2C19 to form p-iodoamphetamine.     

Silica crystals and aluminum salts regulate the production of prostaglandin in macrophages via NALP3 inflammasome-independent mechanisms

Particulates such as silica crystal (silica) and aluminum salts (alum) activate the inflammasome and induce the secretion of proinflammatory cytokines in macrophages. These particulates also induce the production of immunoglobulin E via a T helper 2 (Th2) cell-associated mechanism. However, the mechanism involved in the induction of type 2 immunity has not been elucidated. Here, we showed that silica and alum induced lipopolysaccharide-primed macrophages to produce the lipid mediator prostaglandin E? (PGE?) and interleukin-1β (IL-1β). Macrophages deficient in the inflammasome components caspase 1, NALP3, and ASC revealed that PGE? production was independent of the NALP3 inflammasome. PGE? expression was markedly reduced in PGE synthase-deficient (Ptges?/?) macrophages, and Ptges?/? mice displayed reduced antigen-specific serum IgE concentrations after immunization with alum or silica. Our results indicate that silica and alum regulate the production of PGE? and that the induction of PGE? by particulates controls the immune response in vivo.     

Identification of a novel TPM1 mutation in a family with left ventricular noncompaction and sudden death

Left ventricular noncompaction (LVNC) is a cardiomyopathy morphologically characterized by 2-layered myocardium, numerous prominent trabeculations, and deep intertrabecular recesses communicating with the left ventricular cavity. The purpose of this study was to investigate patients with LVNC for possible disease causing mutations. We screened 4 genes (TAZ, LDB3, DTNA and TPM1) in 51 patients with LVNC for mutations by polymerase chain reaction and direct DNA sequencing. A novel missense substitution in exon 1 of TPM1 (c.109A>G: p.Lys37Glu) was identified in three affected members of a family with isolated LVNC. The substitution brings about a change in amino acid charge at a highly conserved residue and could result in aberrant mRNA splicing. This variant was not identified in 200 normal control samples. Pathologic analysis of a right ventricular myocardial specimen from the proband's maternal aunt revealed endocardial and subendocardial fibrosis with prominent elastin deposition, as well as the presence of adipose tissue between muscle layers, pathologic changes that are distinct from those seen in patients with HCM or DCM. Screening of the proband and her mother for variants in other sarcomeric protein-encoding candidate genes, MYH7, MYBPC3, TNNT2, TNNI3, ACTC, MYL2, and MYL3, did not identify any other non-synonymous variants or variants in splice donor-acceptor sequences that were potentially disease causing. We conclude TPM1 is a potential candidate disease-causing gene for isolated LVNC, especially in patients experiencing sudden death.     

Regulation of osteogenetic differentiation of mesenchymal stem cells by two axial rotational culture

It is crucial to understand how gravitational force affects the osteogenic differentiation of mesenchymal stem cells (MSCs), and these fundamental aspects hold promise for the development of a novel model of MSC regulation for cell proliferation and differentiation. The objective of this study was to investigate how significantly gravitational dispersion affects the spontaneously induced osteogenic differentiation of MSCs. Expression of surface antigen was measured by flow cytometry prior to two axial rotational cultures. About 12,500 hMSC cells were spread on culture wells of 1.8 cm² surface area and incubated for 7 days at 5% CO₂. The culture medium, 10% FCS/DMEM containing 3 ng/ml bFGF, was replaced every 3 days. Four wells then were placed in a 50-ml centrifugal tube filled with 10% FCS/DMEM without bFGF. The centrifugal tube was attached to the center of the rotor, and two axial rotational cultures were started at 10 rpm each of both rotational speeds. It was confirmed that the hMSCs used in this study expressed typical surface antigens as well as a multipotent differentiation ability for either osteogenic or adipogenic differentiation. Spontaneous expression of alkaline phosphatase (Alp) mRNA following the conventional static culture (1G condition) was suppressed by two axial rotational cultures for 7 days (p < 0.05). A separate study indicated that the cell count number eventually increased from 24,700 ± 6,400 to 78,400 ± 18,700 (p < 0.05). In addition, suppressed Alp mRNA was recovered after an additional 7-day culture under static conditions. This result indicated that dispersion of gravity is a promising modality to regulate osteogenic differentiation of hMSCs.     

Influence of pre-laminated material on shock absorption ability in specially designed mouthguard with hard insert and space

Purpose

We have developed a new type of laminated mouthguard, the Hard & Space mouthguard, which incorporates a hard material insert and a space to prevent contact between the mouthguard and the buccal surfaces of the teeth. The purpose of this study was to investigate the effect of this new design on shock absorption.

Methods

Three types of mouthguard (a conventional laminated EVA mouthguard and two 3-layer type “Hard & Space” mouthguards made of 1.8-mm or 3.0-mm thick pre-laminated material (Konbiplast) and EVA with a 1.0-mm space) were impacted. Shock absorption was measured by means of a pendulum type steel ball impact testing machine at impact distances of 10, 20, and 30 cm and a dental study model with strain gauges attached to the lingual surfaces and an accelerometer fixed to the maxilla.

Results

Distortion of the impacted tooth and acceleration of the model were significantly reduced by all types of mouthguard at all 3 impact distances. The effect of the mouthguard was remarkable in terms of tooth distortion: both thicknesses of Hard & Space mouthguard showed more than 90% shock absorption, compared with only approximately 55–78% with an EVA mouthguard. Furthermore, shock absorption with the thicker 3.0-mm Hard & Space mouthguard reached more than 95% at the highest impact power.

Conclusion

Within the limitations of this laboratory study, Hard & Space mouthguards showed significantly greater buffer capacity than a conventional EVA mouthguard in terms of tooth distortion at the 3 impact powers tested.     

Characteristics of hydroxyapatite film formed on human enamel with the powder jet deposition technique

This study aimed to create hydroxyapatite (HAp) film by powder jet deposition with manipulating the blasting nozzle above human enamel and to examine the microstructural and mechanical properties of the HAp film and the bonding strength at the interface between the HAp film and the enamel substrate. HAp particles calcinated at 1200°C with an average size of 4.7 μm were used. The HAp particles were mixed with carrier gas (N?) to form an aerosol flow and was accelerated and blasted from the nozzle onto the enamel substrate at room temperature and atmospheric pressure. To evaluate the microstructure, scanning electron microscope (SEM) images of the surface and cross section of the HAp films and a three-dimensional profile of the HAp films were observed. To evaluate the mechanical properties, the micro-Vickers hardness and the bonding strength of the HAp films to the enamel substrate were measured. The deposition area of the HAp film was over 3 × 4 mm. The average and maximum thickness were about 30 and 40 μm, respectively. No significant difference was observed between the hardness of the HAp film and the enamel (p > 0.05). The bonding strength of the HAp film was the same as the bonding strength between composite resin and enamel. Compared with previous reports, wider and thicker HAp film was created on the enamel substrate successfully. The HAp film, which has same hardness with enamel and same bonding strength to the enamel with composite resin, would be a candidate as dental restorative materials.     

Reevaluation of analytical methods for photogenerated singlet oxygen

The aim of the present study is to compare different analytical methods for singlet oxygen and to discuss an appropriate way to evaluate the yield of singlet oxygen photogenerated from photosensitizers. Singlet oxygen photogenerated from rose bengal was evaluated by electron spin resonance analysis using sterically hindered amines, spectrophotometric analysis of 1,3-diphenylisobenzofuran oxidation, and analysis of fluorescent probe (Singlet Oxygen Sensor Green®). All of the analytical methods could evaluate the relative yield of singlet oxygen. The sensitivity of the analytical methods was 1,3-diphenylisobenzofuran < electron spin resonance < Singlet Oxygen Sensor Green®. However, Singlet Oxygen Sensor Green® could be used only when the concentration of rose bengal was very low (<1 µM). In addition, since the absorption spectra of 1,3-diphenylisobenzofuran is considerably changed by irradiation of 405 nm laser, photosensitizers which are excited by light with a wavelength of around 400 nm such as hematoporphyrin cannot be used in the 1,3-diphenylisobenzofuran oxidation method. On the other hand, electron spin resonance analysis using a sterically hindered amine, especially 2,2,6,6-tetramethyl-4-piperidinol and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide, had proper sensitivity and wide detectable range for the yield of photogenerated singlet oxygen. Therefore, in photodynamic therapy, it is suggested that the relative yield of singlet oxygen generated by various photosensitizers can be evaluated properly by electron spin resonance analysis.