Characterization of human iPSC-derived sensory neurons and their functional assessment using multi electrode array.

Sensory neurons are afferent neurons in sensory systems that convert stimuli and transmit information to the central nervous system as electrical signals. Primary afferent neurons that are affected by non-noxious and noxious stimuli are present in the dorsal root ganglia (DRG), and the DRG sensory neurons are used as an in vitro model of the nociceptive response. However, DRG derived from mouse or rat give a low yield of neurons, and they are difficult to culture. To help alleviate this problem, we characterized human induced pluripotent stem cell (hiPSC) derived sensory neurons. They can solve the problems of interspecies differences and supply stability. We investigated expressions of sensory neuron related proteins and genes, and drug responses by Multi-Electrode Array (MEA) to analyze the properties and functions of sensory neurons. They expressed nociceptor, mechanoreceptor and proprioceptor related genes and proteins. They constitute a heterogeneous population of their subclasses. We confirmed that they could respond to both noxious and non-noxious stimuli. We showed that histamine inhibitors reduced histamine-induced neuronal excitability. Furthermore, incubation with a ProTx-II and Nav1.7 inhibitor reduced the spontaneous neural activity in hiPSC-derived sensory neurons. Their responsiveness was different from each drug. We have demonstrated that hiPSC-derived sensory neurons combined with MEA are good candidates for drug discovery studies where DRG in vitro modeling is necessary.

Enhancement and maintenance of hepatic metabolic functions by controlling 3D aggregation of cryopreserved human iPS cell-derived hepatocyte-like cells.

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells are potent cells to study individual-specific hepatotoxicity for drug screening test. However, the functions of metabolic enzymes are practically low. Here, we reconstituted stable and compact 3D spheroids of commercially available cryopreserved HLCs by our original spheroid formation method with high viscous methylcellulose medium. 3D formation enhanced the hepatic functions and maintained the functions for 14 days. Especially, the expression of cytochrome P450s was 10- to 100-fold enhanced compared to conventional 2D culture, which is applicable to a typical drug-metabolizing test using liquid chromatograph-tandem mass spectrometer. In conclusion, we successfully formed human HLC spheroid from commercially available cryo-preserved cells, which realized remarkable hepatic maturation by prolonged 3D culture, especially in terms of drug-metabolizing enzymes. Our spheroid formation technology has the potential to make HLC spheroids a potent tool in aspects of pharmaceutical research, such as drug screening and pharmacokinetic studies.

PEGylation of silver nanoparticles by physisorption of cyclic poly(ethylene glycol) for enhanced dispersion stability, antimicrobial activity, and cytotoxicity.

Silver nanoparticles (AgNPs) are practically valuable in biological applications. However, no steady PEGylation has been established, which is essential for internal use in humans or animals. In this study, cyclic PEG (c-PEG) without any chemical inhomogeneity is physisorbed onto AgNPs to successfully PEGylate and drastically enhance the dispersion stability against physiological conditions, white light, and high temperature. In contrast, linear HO-PEG-OH and MeO-PEG-OMe do not confer stability to AgNPs, and HS-PEG-OMe, which is often used for gold nanoparticles, sulfidates the surface to considerably degrade the properties. TEM shows an essentially intact nanostructure of c-PEG-physisorbed AgNPs even after heating at 95 °C, while complete disturbance is observed for other AgNPs. Molecular weight- and concentration-dependent stabilization by c-PEG is investigated, and DLS and ζ-potential measurements prove the formation of a c-PEG layer on the surface of AgNPs. Furthermore, c-PEG-physisorbed AgNPs exhibit persistent antimicrobial activity and cytotoxicity.

Topology and Sequence-Dependent Micellization and Phase Separation of Pluronic L35, L64, 10R5, and 17R4: Effects of Cyclization and the Chain Ends.

The topology effects of cyclization on thermal phase transition behaviors were investigated for a series of amphiphilic Pluronic copolymers of both hydrophilic-hydrophobic-hydrophilic and hydrophobic-hydrophilic-hydrophobic block sequences. The dye solubilization measurements revealed the lowered critical micelle temperatures (TCMT) along with the decreased micellization enthalpy (ΔHmic) and entropy (ΔSmic) for the cyclized species. Furthermore, the transmittance and dynamic light scattering (DLS) measurements indicated a block sequence-dependent effect on the clouding phenomena, where a profound decrease in cloud point (Tc) was only found for the copolymers with a hydrophilic-hydrophobic-hydrophilic block sequence. Thus, the effect of cyclization on these critical temperatures was manifested differently depending on its block sequence. Finally, a comparison of the linear hydroxy-terminated, methoxy-terminated, and cyclized species indicated the effect of cyclization to be unique from a simple elimination of the terminal hydrophilic moieties.

Cyclization of PEG and Pluronic Surfactants and the Effects of the Topology on Their Interfacial Activity.

A series of cyclic surfactants were synthesized from a poly(ethylene glycol) (PEG) homopolymer and Pluronic surfactants L35, L64, P123, F68, 10R5, and 17R4, and their interfacial activity depending on the topology, chain ends, and block sequence was investigated. The cyclization was performed in a single step through etherification of the PEG homopolymer and the hydrophilic-hydrophobic-hydrophilic (ABA type) poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PEG-PPG-PEG), while the hydrophobic-hydrophilic-hydrophobic (BAB type) PPG-PEG-PPG was cyclized via acetalization. The cyclized surfactants were rigorously characterized by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Cyclization of the surfactants induced a significant decrease in the hydrodynamic volume, which was more pronounced than that of the PEG homopolymer. Surface tension (γ) measurements indicated that the interfacial activity of the cyclized surfactants is stronger than their corresponding linear precursors, due to the increase in the surfactant density at the air-water interface as a consequence of the decreased molecular occupational area (A) upon cyclization. In the case of the PEG homopolymer, A considerably decreased from 410 Å2 for the linear PEG prepolymer to 100 Å2 for the cyclized PEG product. While the effects of chain-end groups were found to be limited to surfactants of relatively small molecular weights, the influence of cyclization depended strongly on the hydrophilic/hydrophobic ratio; the higher the PEG composition the surfactants had, the larger the decrease in γ and A; in other words, stronger enhancement in the interfacial activity was observed.

Metal-Organic Frameworks for Practical Separation of Cyclic and Linear Polymers.

The purification step in the manufacturing of cyclic polymers is difficult as complete fractionation to eliminate linear impurities requires considerable effort. Here, we report a new polymer separation methodology that uses metal-organic frameworks (MOFs) to discriminate between linear and cyclic polyethylene glycols (PEGs) via selective polymer insertion into the MOF nanopores. Preparation of a MOF-packed column allowed analytical and preparative chromatographic separation of these topologically distinct pairs. In addition, gram-scale PEGs with only cyclic structures were successfully obtained from a crude reaction mixture by using MOF as an adsorbent.

Morphological alterations of periodontal pocket epithelium following Nd:YAG laser irradiation.

OBJECTIVE: The purpose of this in vivo study was to examine morphologic alterations in the periodontal pocket epithelium with presence or absence of clinical inflammation following the use of the Neodymium: Yttrium-Aluminum-Garnet (Nd:YAG) laser irradiation. BACKGROUND DATA: Subgingival Nd:YAG laser irradiation has been proposed as an alternative technique for treatment of chronic periodontitis. Several published studies have reported the clinical outcomes of such treatment. METHODS: Twenty patients, diagnosed with moderate chronic periodontitis, were selected for the study. A total of 32 sites was identified and divided into a control (n=18) and laser-treated test groups (n=14). Probing depth (PD) and bleeding on probing (BOP) were recorded for all sites. Test sites were irradiated with an Nd:YAG laser using parameters of 2 W, 200 mJ pulse energy, and 10 pps delivered through a 320 µm diameter tip. Total laser treatment time ranged from 1 to 2 min. Following treatment, all specimens were harvested via biopsy and processed for scanning electron microscopy (SEM) and histologic examination. RESULTS: Control group specimens, depending upon initial PD, exhibited either a relatively smooth and intact epithelium with little desquamation (PD≤3 mm), or increasing degrees of epithelial desquamation and leukocytic infiltration at a PD of ≥4 mm. In the laser-treated test group, the specimens with PD≤3 mm that were BOP negative (-) exhibited a thin layer of epithelium that was disrupted. In the specimens with initial PD of ≥4 mm, complete removal of the epithelium whose extent and degree were increasing, was observed in the inflamed portion, while epithelium remained in the uninflamed portion. CONCLUSIONS: The SEM and histologic findings demonstrated the feasibility of ablating pocket epithelium with an Nd:YAG laser irradiation using parameters of 2 W of power (200 mJ, 10 pps). Furthermore, the presence or absence of clinical inflammation appeared to have an impact on the degree of laser-mediated epithelial ablation.

Identification of an HLA-A2-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2).

We herein report the identification of an HLA-A2 supertype-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2), which is known to be a diagnostic marker and a potential therapeutic target for renal cell carcinoma. Among several candidate peptides predicted by the HLA-binding prediction algorithm, HIG2-9-4 peptide (VLNLYLLGV) was able to effectively induce peptide-specific cytotoxic T lymphocytes (CTLs). The established HIG2-9-4 peptide-specific CTL clone produced interferon-γ (IFN-γ) in response to HIG2-9-4 peptide-pulsed HLA-A*02:01-positive cells, as well as to cells in which HLA-A*02:01 and HIG2 were exogenously introduced. Moreover, the HIG2-9-4 peptide-specific CTL clone exerted cytotoxic activity against HIG2-expressing HLA-A*02:01-positive renal cancer cells, thus suggesting that the HIG2-9-4 peptide is naturally presented on HLA-A*02:01 of HIG-2-expressing cancer cells and is recognized by CTLs. Furthermore, we found that the HIG2-9-4 peptide could also induce CTLs under HLA-A*02:06 restriction. Taken together, these findings indicate that the HIG2-9-4 peptide is a novel HLA-A2 supertype-restricted epitope peptide that could be useful for peptide-based immunotherapy against cancer cells with HIG2 expression.

Nd:YAG laser irradiation of the tooth root surface inhibits demineralization and root surface softening caused by minocycline application.

OBJECTIVE: The aim of this study was to investigate the effect of laser irradiation on root surface demineralization caused by local drug delivery systems (DDS), and to evaluate the effect of sealing on drug retention. BACKGROUND DATA: The duration of supportive periodontal treatment (SPT) has increased with increasing life expectancy. Repeated root planing and DDS application during SPT should be reconsidered with regard to their effects on the root surface. METHODS: Extracted human teeth were collected, cut into 3 × 3 × 2 mm root dentin specimens, and divided randomly into eight groups with various combinations of Nd:YAG laser power (0, 0.5, 1, and 2 W), with and without DDS (minocycline HCl). Specimen microhardness and calcium (Ca) solubility were measured after treatment. The specimens (control and laser and DDS groups) were examined by scanning electron microscopy. Forty SPT patients were recruited, to assess the effect of periodontal pocket sealing on drug retention. RESULTS: Laser irradiation increased the microhardness of root specimens in an energy-dependent manner. Calcium solubilities decreased from the 0 W+DDS group to the 2.0 W+DDS group. The mean Ca solubilities in the 1.0 W+DDS and 2.0 W+DDS groups were significantly lower than in the 0 W+DDS group (p<0.01, p<0.001, respectively). Laser irradiation counteracted the softening effect of DDS. Morphologic change was observed in the 2 W+DDS group; however, no morphologic changes were observed in the control and the 1 W+DDS groups. The mean concentration of minocycline in the periodontal pocket 24 h after application was 252.79 ± 67.50 µg/mL. CONCLUSIONS: Laser irradiation of the root surface inhibited the softening and decalcification caused by minocycline HCl. Sealing the periodontal pockets effectively improved drug retention. These results suggest that the combination of laser irradiation and DDS could benefit patients receiving repeated SPT.

Identification of HLA-A24-restricted novel T Cell epitope peptides derived from P-cadherin and kinesin family member 20A.

We here identified human leukocyte antigen-(HLA-)A(∗)2402-restricted epitope peptides from Cadherin 3, type 1, P-cadherin (CDH3) and kinesin family member 20A (KIF20A) that were found to be specifically expressed in cancer cells through genome-wide expression profile analysis. CDH3-10-807 peptide and KIF20A-10-66 peptide successfully induced specific CTL clones, and these selectively responded to COS7 cells expressing both HLA-A(∗)2402 and respective protein while did not respond to parental cells or COS7 cells expressing either HLA-A(∗)2402 or respective protein. Furthermore, CTL clones responded to cancer cells that endogenously express HLA-A(∗)2402 and respective protein, suggesting that CDH3-10-807 peptide and KIF20A-10-66 peptide are naturally presented on HLA-A(∗)2402 molecule of human cancer cells. Our results demonstrated that CDH3-10-807 peptide and KIF20A-10-66 peptide are novel HLA-A24-restricted tumor-associated antigens and would be applicable for CTL-inducing cancer therapies.

Effects of Er,Cr:YSGG laser irradiation on the root surface: morphologic analysis and efficiency of calculus removal.

BACKGROUND: This in vitro study was performed to determine the appropriate power output setting for an erbium, chromium-doped:yttrium, scandium, gallium, and garnet (Er,Cr:YSGG) laser used in periodontal pocket irradiation by examining the morphologic alterations of the root surfaces and the efficiency of calculus removal. METHODS: Sixty-five non-carious extracted human teeth were used in this study. For morphologic analysis of the root surface, the clean, single roots of 22 teeth were separated into 91 pieces, and these pieces were immersed in acrylic resin. The specimens with root-surface exposure were prepared and divided randomly into three groups: a control group (N=8), an irradiation without water group (no water [NW] group; N=39), and an irradiation in water to simulate the conditions in a periodontal pocket group (in water [IW] group; N=44). The power output settings for laser irradiation were 0.5, 1.0, 1.5, and 2.0 W for each group. The roughness (Ra), depth (Z), and width (X) of the disk specimens were determined after laser irradiation. Eight other single-rooted teeth were examined by scanning electron microscopy (SEM) after laser irradiation under the same conditions. Thirty-five single- or multirooted teeth with heavy subgingival calculus were used to test the efficiency of laser scaling. The efficiency of calculus removal was quantified by measuring the time needed to remove the calculus completely using the laser. RESULTS: The mean Ra and Z values in the IW group were significantly higher than in the NW group with the same power output. In addition, these values with 0.5- and 1.0-W power output settings were significantly lower than with 1.5- and 2.0-W settings in the NW and IW groups. No obvious morphologic differences could be found between the 0.5- and 1.0-W power output specimens under SEM. Additionally, thermal alterations, i.e., carbonization or melting, were completely absent in the IW group. Regarding the efficiency of calculus removal, the 0.5-W setting (0.11+/-0.036 mm2/second) was significantly inferior to the 1.0-W setting (0.27+/-0.043 mm2/second). However, there was no significant difference between 1.0- and 1.5-W (0.36+/-0.11 mm2/second). The 2.0-W setting (0.63+/-0.272 mm2/second) was much more efficient but resulted in significant morphologic alterations. CONCLUSIONS: Based on these findings, it is appropriate to use a 1.0-W power output setting with an Er,Cr:YSGG laser for root scaling. This may be done without any conspicuous morphologic alterations to the root surface and with acceptably efficient removal of calculus.

Linkage mapping of AFLP and microsatellite DNA markers with the body color- and sex-determining loci in the guppy (Poecilia reticulata).

The guppy is an ornamental fish species that exhibits various phenotypic characteristics, such as body color and fin-shape. Although linkage relationships of a limited number of phenotypic traits have already been investigated, the association between phenotypic and molecular markers is still unknown. We constructed a total of 35 linkage groups for the guppy using 186 polymorphic loci of AFLP and microsatellite DNA. The locus related to the yellow body color was linked with ten markers and the sex-determination locus was linked with five markers.

Preliminary study on linkage mapping based on microsatellite DNA and AFLP markers using homozygous clonal fish in ayu (Plecoglossus altivelis).

A mapping referential family (F(1)) of ayu was produced by crossing a normal diploid male with a homozygous clonal female. A genetic linkage map was constructed using 191 amplified fragment length polymorphism (AFLP) and 4 microsatellite DNA markers. A total of 178 loci were mapped in 36 linkage groups comprising 1659.6 cM, which includes approximately 77.3% to 81.8% of the total genome. As the markers were randomly distributed over the genome, they showed high efficiency for the construction of a wide linkage map.