Phenotype-genotype relationships in Xenopus sox9 crispants provide insights into campomelic dysplasia and vertebrate jaw evolution.

Since CRISPR-based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non-syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame-shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in-frame deletion mutations expected to delete part of the jawed vertebrate-specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype-genotype relationships behind syndromic diseases and examining the tissue-specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution.

The thermosensitive TRPV3 channel contributes to rapid wound healing in oral epithelia.

The oral cavity provides an entrance to the alimentary tract to serve as a protective barrier against harmful environmental stimuli. The oral mucosa is susceptible to injury because of its location; nonetheless, it has faster wound healing than the skin and less scar formation. However, the molecular pathways regulating this wound healing are unclear. Here, we show that transient receptor potential vanilloid 3 (TRPV3), a thermosensitive Ca(2+)-permeable channel, is more highly expressed in murine oral epithelia than in the skin by quantitative RT-PCR. We found that temperatures above 33°C activated TRPV3 and promoted oral epithelial cell proliferation. The proliferation rate in the oral epithelia of TRPV3 knockout (TRPV3KO) mice was less than that of wild-type (WT) mice. We investigated the contribution of TRPV3 to wound healing using a molar tooth extraction model and found that oral wound closure was delayed in TRPV3KO mice compared with that in WT mice. TRPV3 mRNA was up-regulated in wounded tissues, suggesting that TRPV3 may contribute to oral wound repair. We identified TRPV3 as an essential receptor in heat-induced oral epithelia proliferation and wound healing. Our findings suggest that TRPV3 activation could be a potential therapeutic target for wound healing in skin and oral mucosa.

Membrane Translocation and Organelle-Selective Delivery Steered by Polymeric Zwitterionic Nanospheres.

The majority of nanoparticles designed for cellular delivery of drugs and imaging agents enter the cell via endocytotic pathways leading to their entrapment in endosomes that present a robust barrier to further trafficking of the nanoparticles within the cells. A few materials, such as the cell penetrating peptides (CPPs), are known to enter cells not only via endocytosis, but also via translocation through the cell membrane into the cytoplasm, successfully bypassing the endosomes. We report here that random copolymers of 3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate and poly(ethylene glycol) methacrylate, p(DMAPS-ran-PEGMA), are internalized in cells primarily via translocation through the cell membrane rather than endocytosis. The properties of the polymers and their modes of uptake were investigated systematically by dynamic light scattering, confocal fluorescence microscopy, and flow cytometry. Using specific inhibitors of the cellular uptake machinery in a human cervical carcinoma cell line (HeLa), we show that these nontoxic synthetic polyzwitterions exist in cell media as self-assembled nanospheres that unravel as they adsorb on the plasma membrane and translocate through it. Conjugates of p(DMAPS-ran-PEGMA) with rhodamine B were delivered selectively to the mitochondria, whereas doxorubicin (Dox)-p(DMAPS-ran-PEGMA) conjugates were accumulated in both the nucleus and the mitochondria, effectively inducing apoptosis in HeLa cells. These findings suggest that the noncytotoxic and readily synthesized p(DMAPS-ran-PEGMA) can find applications as bioimaging tools and drug nanocarriers.

Self-assembled microspheres driven by dipole-dipole interactions: UCST-type transition in water.

A double hydrophilic block copolymer, poly(ethylene glycol)-poly(3-dimethyl (methacryloyloxyethyl) ammonium propane sulfonate) (PEG-SB), is synthesized by reversible addition-fragmentation transfer (RAFT) polymerization using PEG methyl ether (4-cyano-4-pentanoate dodecyl trithiocarbonate) as a chain transfer agent. PEG-SB forms multi-layered microspheres with dipole-dipole interactions of the SB side chains as the driving force. The PEG-SB polymers show an upper critical solution temperature (UCST) and the UCST is controllable by the polymerization degree. The PEG-SB microspheres are dissociated above the UCST and then monodispersed microspheres (∼1 µm) are obtained when the solution temperature is decreased below the UCST again. The disassociation/association of the microspheres is also controllable using the concentration of NaCl. These multi-responsive microspheres could be a powerful tool in the field of nano-biotechnology.

Evaluation of optimal length and insertion torque for miniscrews.

INTRODUCTION: The purpose of this article was to test the theory that short miniscrews will decrease the possibility of damaging the root, but the failure rate will increase. METHODS: One hundred eighty-six miniscrews (diameter, 1.3 × 5 mm, n = 63; 6 mm, n = 62; 7 mm, n = 61) were placed in 105 consecutive patients. Multislice computed tomography and cone-beam computed tomography scans were taken before and after miniscrew placement. Insertion torque was measured at miniscrew placement. RESULTS: The success rate of the miniscrews in the maxilla (93.4%) was higher than that in the mandible (70.3%). A significantly lower success rate with 5-mm miniscrews was observed compared with 6-mm and 7-mm miniscrews in the mandible. Miniscrews placed in less than approximately 3.8 mm of bone and those within 1.4 mm of the root had significantly higher failure rates. Miniscrews placed with insertion torque greater than 10 Ncm had a tendency for a lower success rate. CONCLUSIONS: The optimum lengths of miniscrews of a diameter of 1.3 mm are 5 mm in the maxilla and 6 mm in the mandible. They should be placed at a distance from the root with insertion torque less than 10 Ncm for safe orthodontic anchorage without failure.

Hexagonally Packed Cylindrical Structures with Multiple Satellites from Pentablock Quarterpolymers of the AB1CB2D Type and Their Blends with Homopolymers.

Three kinds of hexagonally packed cylindrical (HPC) structures with 10/8 satellites have been found from a neat pentablock quarterpolymer of the AS1IS2P type, composed of poly(4-vinylbenzyldimethylamine) (A), polystyrene (S), polyisoprene (I), and poly(2-vinylpyridine) (P), and block polymer/homopolymer blends. AS1IS2P-2 (M = 276k, ϕA/ϕS1/ϕI/ϕS2/ϕP = 0.08/0.17/0.12/0.33/0.30) shows HPC of P with 10 single-arrayed I satellites and 6 A subsatellites in the S matrix, where the I/P ratio number is 5, while AS1IS2P-2 (M = 325k, ϕA/ϕS1/ϕI/ϕS2/ϕP = 0.07/0.31/0.14/0.18/0.30)/Sh(M;11k) = 90/10 blend reveals another HPC of P with 10 I satellites, giving an I/P ratio of 6, whose array of I is a single/double mixture. Moreover, AS1IS2P-1/Sh/Ah(M;9k) = 90/5/5 blend represents two enantiomeric HPC packings with 8 double-arrayed I satellites having an I/P ratio of 7, which can be considered to be approximants of two enantiomeric 3.3.3.3.6 Archimedean tilings. These structures were conceived to be formed by the enhanced miscibility of tail A-chains with added homopolymers, resulting in an increase of the (A + S)/I interaction leading to the split of I domains.

Fast and effective mitochondrial delivery of ω-Rhodamine-B-polysulfobetaine-PEG copolymers.

Mitochondrial targeting and entry, two crucial steps in fighting severe diseases resulting from mitochondria dysfunction, pose important challenges in current nanomedicine. Cell-penetrating peptides or targeting groups, such as Rhodamine-B (Rho), are known to localize in mitochondria, but little is known on how to enhance their effectiveness through structural properties of polymeric carriers. To address this issue, we prepared 8 copolymers of 3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate and poly(ethyleneglycol) methacrylate, p(DMAPS-ran-PEGMA) (molecular weight, 18.0 < M n < 74.0 kg/mol) with two different endgroups. We labeled them with Rho groups attached along the chain or on one of the two endgroups (α or ω). From studies by flow cytometry and confocal fluorescence microscopy of the copolymers internalization in HeLa cells in the absence and presence of pharmacological inhibitors, we established that the polymers cross the cell membrane foremost by translocation and also by endocytosis, primarily clathrin-dependent endocytosis. The most effective mitochondrial entry was achieved by copolymers of M n < 30.0 kg/mol, lightly grafted with PEG chains (< 5 mol %) labeled with Rho in the ω-position. Our findings may be generalized to the uptake and mitochondrial targeting of prodrugs and imaging agents with a similar polymeric scaffold.

Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis.

In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined. Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains. It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain. Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain. On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant. The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.

Formulation study and drug release mechanism of a new theophylline sustained-release preparation.

Two matrix theophylline tablets with different release mechanisms were compared. Tablet A was a swelling/disintegration-type wax matrix made of hydrophobic wax granules, consisting of stearic acid, hydrogenated oil and glycerol esters of fatty acids, and hydrophilic polymer granules composed primarily of hydroxypropyl methylcellulose (HPMC). We named Tablet A the cluster tablet. Tablet B was a gel matrix made of hydrophobic ethylcellulose granules, consisting of ethylcellulose and hydrogenated oil, and hydrophilic polymer granules consisting of HPMC and hydroxylpropylmethylcellulose acetate succinate (HPMCAS). The formulations were screened in vitro according to their dissolution characteristics. The drug release from each preparation was analyzed using release kinetics theories. In Tablet A, the value of the exponent(n) representing the apparent diffusion mechanism determined from the Korsmeyer-Peppas model equation was about 0.6 and was unlikely to be affected by the rotation speed. In Tablet B, the value of the exponent(n) by the Korsmeyer-Peppas model equation changed with the paddle rotation speed. These results suggested that the drug release mechanism of Tablet B is greatly affected by the extent of physical force in the gastrointestinal tract.

Tactile texture and friction of soft sponge surfaces.

We evaluated the tactile texture and frictional properties of five soft sponges with various cell sizes. The frictional forces were measured by a friction meter containing a contact probe with human-finger-like geometry and mechanical properties. When the subjects touched these sponges with their fingers, hard-textured sponges were deemed unpleasant. This tactile feeling changed with friction factors including friction coefficients, their temporal patterns, as well as mechanical and shape factors. These findings provide useful information on how to control the tactile textures of various sponges.

Trading polymeric microspheres: exchanging DNA molecules via microsphere interaction.

A new class of artificial molecular transport system is constructed by polymeric microspheres. The microspheres are prepared by self-assembly of poly(ethylene glycol)-block-poly(3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate), PEG-b-PDMAPS, by intermolecular dipole-dipole interaction of sulfobetaine side chains in water. Below the upper critical solution temperature (UCST) of PEG-b-PDMAPS, the microspheres (∼1µm) interact with other microspheres by partial and transit fusion. In order to apply the interaction between microspheres, a 3'-TAMRA-labeled single-stranded DNA oligomer (ssDNA) is encapsulated into a PEG-b-PDMAPS microsphere by thermal treatment. The exchange of ssDNA between microspheres is confirmed by fluorescence resonance energy transfer (FRET) quenching derived from double-stranded formation with complementary 5'-BHQ-2-labeled ssDNA encapsulated in PEG-b-PDMAPS microspheres. The exchange rate of ssDNA is controllable by tuning the composition of the polymer. The contact-dependent transport of molecules can be applied in the areas of microreactors, sensor devices, etc.

Analysis of pain level in cases treated with Invisalign aligner: comparison with fixed edgewise appliance therapy.

BACKGROUND: The aim of this study was to evaluate and compare the difference in the level of pain using the visual analog scale (VAS) between cases treated with the edgewise appliance and Invisalign. In addition, the cause of pain and discomfort in the Invisalign cases was identified. METHODS: The sample consisted of 145 cases for the edgewise group (EG; n=55), Invisalign group (IG; n=38), and edgewise and Invisalign group (EIG; n=52). VAS scores were collected during the first three stages (first stage: 0 to 7 days, second stage: 14 to 21 days, and third stage: 28 to 35 days) and at the end of the treatment (overall VAS score). Evaluation of the cause of pain was categorized into three different types of problem (category 1: non-smoothed marginal ridge or missing materials, category 2: deformation of attachments, and Category 3: deformation of the tray). Statistical comparison of VAS scores between groups was performed by two-way analysis of variance. RESULTS: A significantly higher VAS score was observed at 3 and 4 days after, at 1, 2, and 3 days after, and at 2 and 3 days after in stages 1, 2, and 3, respectively, in EG compared to EIG and IG. A significant difference was observed in overall VAS scores between EG and IG in intensity of pain, number of days that pain lasted, and discomfort level. Only intensity of pain resulted in a significant difference between EG and EIG. Most of the causes of problem in the Invisalign cases were deformation of the tray. CONCLUSIONS: Invisalign may offer less pain compared to the edgewise appliance during the initial stages of treatment. In the use of Invisalign, deformation of tray must be carefully checked to avoid pain and discomfort for the patients.

Temperature-responsive telechelic dipalmitoylglyceryl poly(N-isopropylacrylamide) vesicles: real-time morphology observation in aqueous suspension and in the presence of giant liposomes.

Telechelic α,ω-di(twin-tailed poly(N-isopropylacrylamides)) form polymersomes in water that increase in size by fusion when the water temperature exceeds the polymers cloud point temperature. Hybrid vesicles form in mixed suspensions of giant phospholipid liposomes and polymersomes by adsorption/fusion, and undergo further transformations, such as fission.

Efficacy and safety of additional 200-mg dose of celecoxib in adult patients with postoperative pain following extraction of impacted third mandibular molar: a multicenter, randomized, double-blind, placebo-controlled, phase II study in Japan.

BACKGROUND: Although third mandibular molar extraction is a widely used and validated model of acute pain for evaluating analgesic efficacy, a large proportion of patients experience moderate or severe pain following this procedure and require analgesia. Current treatment options have been associated with safety concerns and alternative therapies are sought. OBJECTIVE: Our aim was to assess the efficacy and safety of an additional 200-mg dose of celecoxib, administered 5 to 12 hours after an initial 400-mg dose of the drug for the treatment of moderate or severe acute pain following extraction of an impacted third mandibular molar. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled, Phase II study. Patients experiencing moderate or severe pain within 1 to 2 hours following extraction of an impacted third mandibular molar received an initial 400-mg dose of celecoxib. Patients requiring additional analgesia were subsequently randomized to receive either an additional 200-mg dose of celecoxib or placebo 5 to 12 hours after the initial dose. The study was designed and conducted by Pfizer Inc. for approval of celecoxib in Japan for the indication of acute pain. The primary end point was the patient's impression of efficacy (4-category global evaluation scale). Secondary efficacy end points included pain intensity on a 4-category pain intensity scale, pain intensity on a 100-mm visual analog scale (VAS), and the pain intensity difference (100-mm VAS). In an exploratory analysis, use of rescue medication was evaluated. Primary and secondary end points were analyzed using the full analysis set. Assessment of the safety profile included a physical examination, measurement of pulse rate and blood pressure, standard 12-lead ECG, and laboratory tests. RESULTS: A total of 69 patients (celecoxib, 42/64 [65.6%]; placebo, 27/58 [46.6%]) received the additional dose of study medication; all completed the study without the need for rescue medication. A significantly higher proportion of patients in the celecoxib 200 mg group (41/64 [64.1%]) compared with the placebo group (15/58 [25.9%]) rated the study medication as "good" or "excellent" ≥ 2 hours after the additional dose (P < 0.0001). Pain intensity (VAS) 2 hours after the additional dose was significantly higher in the placebo group than in the celecoxib 200 mg group (P = 0.0003). The reduction in pain intensity from baseline to 2 hours after the additional dose of study medication was also significantly greater in the celecoxib 200 mg group than in the placebo group (P < 0.0001). The incidence of treatment-related, all-cause adverse events was slightly lower in patients receiving celecoxib 200 mg (20.3%) compared with placebo (31.0%). CONCLUSIONS: Overall, an additional 200-mg dose of celecoxib was well tolerated and efficacious in reducing the pain associated with extraction of an impacted third mandibular molar in the study population. ClinicalTrials.gov identifier: NCT01062113.

Extracellular matrix remodeling in oral submucous fibrosis: its stage-specific modes revealed by immunohistochemistry and in situ hybridization.

BACKGROUND: Oral submucous fibrosis (OSF) is a chewing habit-related pre-cancerous condition of the oral mucosa affecting predominantly south Asians. It is histopathologically characterized by epithelial atrophy and fibrosis of the subepithelial connective tissue. Fibrosis extends all the way into the muscle layer, leading to difficulty in mouth opening. However, the dynamics of extracellular matrix (ECM) remodeling with OSF progression is largely unknown. METHODS: Forty biopsy specimens of OSF and 10 of normal buccal mucosa were examined for expression/deposition modes of eight ECM molecules by histochemistry, immunohistochemistry, and in situ hybridization. RESULTS: In the early stage of OSF, tenascin, perlecan, fibronectin, collagen type III were characteristically enhanced in the lamina propria and the submucosal layer. In the intermediate stage, the ECM molecules mentioned above and elastin were extensively and irregularly deposited around muscle fibers. In the advanced stage, such ECM depositions decreased and were entirely replaced with collagen type I only. Their gene expression levels varied with progression of fibrosis, but the mRNA signals were confirmed in fibroblasts in the submucosal fibrotic areas. CONCLUSIONS: The results indicate that the ECM remodeling steps in OSF are similar to each phase of usual granulation tissue formation. Restricted mouth opening may be a result of loss of variety of ECM molecules including elastin into the homogeneity of collagen type I replacing muscle fibers.

Cooperative structural change of actin filaments interacting with activated myosin motor domain, detected with copolymers of pyrene-labeled actin and acto-S1 chimera protein.

Acto-S1 chimera proteins CP24 and CP18 carry the entire actin sequence, inserted in loop 2 of the motor domain of Dictyostelium myosin II, and have MgATPase activity close to that of natural Dictyostelium actomyosin [M.S.P. Siddique, T. Miyazaki, E. Katayama, T.Q.P. Uyeda, M. Suzuki, Evidence against essential roles of subdomain 1 of actin in actomyosin sliding movements, Biochem. Biophys. Res. Commun. 332 (2005) 474-481]. Here, we examined and detected cooperative structural change of actin filaments accompanying interaction with myosin motor domain in the presence of ATP using copolymer filaments consisting of pyrene-labeled skeletal actin (SA) and either CP24 or CP18. Upon addition of ATP, the fluorescence intensity increased over the range from 380 to 480nm using 365-nm excitation. The relative increases of fluorescence intensity at 390nm were 14%, 46%, and 77% for the copolymer filaments with the CP24 to actin molar ratios of 0.0625, 0.143, and 0.333, respectively, and demonstrated a sigmoid behavior. Stoichiometric analysis indicates that each CP24 molecule appears to affect four actin molecules, on average, in SA-CP24 copolymers, and each CP18 molecule appears to affect three actin molecules in SA-CP18 copolymers.