Tissue-Targeted Transcriptomics Reveals SEMA3D Control of Hypoglossal Nerve Projection to Mouse Tongue Primordia.

The mouse hypoglossal nerve originates in the occipital motor nuclei at embryonic day (E)10.5 and projects a long distance, reaching the vicinity of the tongue primordia, the lateral lingual swellings, at E11.5. However, the details of how the hypoglossal nerve correctly projects to the primordia are poorly understood. To investigate the molecular basis of hypoglossal nerve elongation, we used a novel transcriptomic approach using the ROKU method. The ROKU algorithm identified 3825 genes specific for lateral lingual swellings at E11.5, of which 34 genes were predicted to be involved in axon guidance. Ingenuity Pathway Analysis-assisted enrichment revealed activation of the semaphorin signaling pathway during tongue development, and quantitative PCR showed that the expressions of Sema3d and Nrp1 in this pathway peaked at E11.5. Immunohistochemistry detected NRP1 in the hypoglossal nerve and SEMA3D as tiny granules in the extracellular space beneath the epithelium of the tongue primordia and in lateral and anterior regions of the mandibular arch. Fewer SEMA3D granules were localized around hypoglossal nerve axons and in the space where they elongated. In developing tongue primordia, tissue-specific regulation of SEMA3D might control the route of hypoglossal nerve projection via its repulsive effect on NRP1.

Antioxidants encapsulated milk-derived exosomes for functional food development.

Reactive oxygen species are known to be involved in various diseases, and antioxidant ingredients are expected to essentially prevent diseases and contribute to improving health. However, antioxidants are easily degraded by enzymes before being absorbed in the intestine, so a means of transport that prevents their degradation in the body is necessary. Exosomes, which play an important role in communication between individual cells, have attracted attention as a new transport carrier of miRNA and DNA, but not yet fully exploited in food research. More recently, exosomes extracted from bovine milk began to be widely used as a cost-effective transport carrier not in clinical medicine but also in functional food materials. To develop practical applications as carriers for functional foods, systematic studies are necessary to clarify the introduction efficiency and the properties of encapsulated substances. In this study, we applied electroporation and incubation to encapsulate antioxidants into the exosomes and studied the encapsulation efficiency into the exosomes and the anticancer activity.

Nuclear factor 1 X-type-associated regulation of myogenesis in developing mouse tongue.

OBJECTIVES: The tongue contains skeletal myofibers that differ from those in the trunk, limbs, and other orofacial muscles. However, the molecular basis of myogenic differentiation in the tongue muscles remains unclear. In this study, we conducted comprehensive gene expression profiling of the developing murine tongue. METHODS: Tongue primordia were dissected from mouse embryos at embryonic day (E)10.5-E18.5, while myogenic markers were detected via microarray analysis and quantitative polymerase chain reaction (PCR). In addition to common myogenic regulatory factors such as Myf5, MyoD, myogenin, and Mrf4, we focused on Nfix, which acts as a unique molecular switch triggering the shift from embryonic to fetal myoblast lineage during limb myogenesis. Nfix inhibition was performed using a specific antisense oligonucleotide in the organ culture of tongue primordia. RESULTS: Microarray and ingenuity pathway analyses confirmed the significant upregulation of myogenic signaling molecules, including Nfix, associated with the differentiation of myoblasts from myogenic progenitor cells during E10.5-E11.5. Quantitative PCR confirmed that Nfix expression started at E10.5 and peaked at E14.5. Fetal myoblast-specific genes, such as Mck and Myh8, were upregulated after E14.5, whereas embryonic myoblast-specific genes, such as Myh3 and Myh7, were downregulated. When Nfix was inhibited in the organ culture of tongue primordia, subtle morphological differences were noted in the tongue. Such an observation was only noted in the cultures of E10.5-derived tongue primordia. CONCLUSIONS: These results reveal the contribution of Nfix to tongue myogenesis. Nfix expression during early tongue development may play a vital role in tongue muscle development.

The 1.3-Å resolution structure of bovine cytochrome c oxidase suggests a dimerization mechanism.

Cytochrome c oxidase (CcO) in the respiratory chain catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme and proton pumping across the membrane. Although the functional unit of CcO is monomeric, mitochondrial CcO forms a monomer and a dimer, as well as a supercomplex with respiratory complexes I and III. A recent study showed that dimeric CcO has lower activity than monomeric CcO and proposed that dimeric CcO is a standby form for enzymatic activation in the mitochondrial membrane. Other studies have suggested that the dimerization is dependent on specifically arranged lipid molecules, peptide segments, and post-translationally modified amino acid residues. To re-examine the structural basis of dimerization, we improved the resolution of the crystallographic structure to 1.3 Å by optimizing the method for cryoprotectant soaking. The observed electron density map revealed many weakly bound detergent and lipid molecules at the interface of the dimer. The dimer interface also contained hydrogen bonds with tightly bound cholate molecules, hydrophobic interactions between the transmembrane helices, and a Met-Met interaction between the extramembrane regions. These results imply that binding of physiological ligands structurally similar to cholate could trigger dimerization in the mitochondrial membrane and that non-specifically bound lipid molecules at the transmembrane surface between monomers support the stabilization of the dimer. The weak interactions involving the transmembrane helices and extramembrane regions may play a role in positioning each monomer at the correct orientation in the dimer.

Horizontal Saccadic Velocity in Patients with Exotropia before and after Unilateral Resection and Recession Surgery.

PURPOSE: The effects of strabismus surgery on eye movement are not known in detail, as few studies have compared saccade velocities before and after strabismus surgery. In this study, horizontal saccades were recorded using an eye-tracker in patients with only exotropia to compare the peak velocities (PVs), before and after undergoing strabismus surgery of the same type (unilateral resection and recession). METHODS: Horizontal saccades of monocular vision were recorded using an eye-tracking device in 18 patients with exotropia and 20 normal subjects. All patients were examined using the same method after strabismus surgery. RESULTS: The PVs of adduction and abduction in the patients were higher than those in the normal subjects (in dominant eye, P=0.032 for adduction and P=0.049 for abduction; in nondominant eye, P=0.016 for adduction and P=0.037 for abduction). Following the surgery, the PVs of abduction of the surgical eye (nondominant eye) decreased to the level of the normal subjects (P=0.016). However, there were no correlations between changes in the PVs and the extent of surgery (resection and recession). CONCLUSION: Strabismus surgery normalized the patient's increased PV in the operated eye for abduction of horizontal saccade. Not only peripheral (extraocular muscle) but also central sensory-motor mechanisms may be involved in the changes in PV of horizontal saccades, both of which could result from the improvement of the primary eye position.

Skeletal Rearrangement of Twisted Polycyclic Aromatic Hydrocarbons under Scholl Reaction Conditions.

Treatment of a twisted polycyclic aromatic hydrocarbon containing cyclooctatetraene fused by two 9,9'-bifluorenylidene units under the Scholl reaction conditions (FeCl3 or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and scandium trifluoromethanesulfonate) led to stepwise skeletal rearrangements to afford initially a hydrocarbon with a seven-membered ring and then tetrabenzo[a,d,j,m]coronene with all six-membered rings. The course of the rearrangement was interpreted in terms of the acid-catalyzed isomerization of 9,9'-bifluorenylidene into dibenzo[g,p]chrysene moieties on the basis of theoretical investigations.

A unique database for gathering data from a mobile app and medical prescription software: a useful data source to collect and analyse patient-reported outcomes of depression and anxiety symptoms.

A unique database named 'AN-SAPO' was developed by Iwato Corp. and Japan Brain Corp. in collaboration with the psychiatric clinics run by Himorogi Group in Japan. The AN-SAPO database includes patients' depression/anxiety score data from a mobile app named AN-SAPO and medical records from medical prescription software named 'ORCA'. On the mobile app, depression/anxiety severity can be evaluated by answering 20 brief questions and the scores are transferred to the AN-SAPO database together with the patients' medical records on ORCA. Currently, this database is used at the Himorogi Group's psychiatric clinics and has over 2000 patients' records accumulated since November 2013. Since the database covers patients' demographic data, prescribed drugs, and the efficacy and safety information, it could be a useful supporting tool for decision-making in clinical practice. We expect it to be utilised in wider areas of medical fields and for future pharmacovigilance and pharmacoepidemiological studies.

Fixation stability of the upward gaze in patients with myasthenia gravis: an eye-tracker study.

OBJECTIVE: To quantify fixation stability of the upward gaze in patients with myasthenia gravis (MG) using an eye tracker. METHODS AND ANALYSIS: In this study, 21 normal subjects, 5 patients with MG with diplopia, 5 patients with MG without diplopia and 6 patients with superior oblique (SO) palsy were included. Subjects fixated on a target in the upward direction for 1 min. The horizontal (X) and vertical (Y) eye positions were recorded using an eye tracker. Fixation stability was first quantified using the bivariate contour ellipse areas (BCEA) of fixation points as an index of whole stability. Then, the SDs of the X and Y eye positions (SDX and SDY, respectively) were quantified as indices of directional stability, with the data divided into three 20 s fractions to detect temporal fixation fluctuation. RESULTS: BCEAs were larger in patients with MG (both with and without diplopia) than normal subjects and patients with SO palsy, without significant differences among the three 20 s fractions. Compared with normal subjects, SDXs were larger only in patients with MG with diplopia; SDYs were larger in both patients with MG with and without diplopia. In addition, SDYs in patients with MG with diplopia were larger than those in patients with MG without diplopia and patients with SO palsy. Furthermore, a significant difference among the three 20 s fractions was detected for SDYs in patients with MG with diplopia. CONCLUSION: Patients with MG, especially those with diplopia, exhibit fixation instability in the upward gaze. Non-invasive quantification of fixation stability with an eye tracker is useful for precisely identifying MG-specific fatigue characteristics. TRIAL REGISTRATION NUMBER: UMIN000023468; pre-results.

Diindenopyrenes: Extended 1,6- and 1,8-Pyrenoquinodimethanes with Singlet Diradical Characters.

Diindenopyrene 4b and its diastereomer, which are extended homologues of 1,6- and 1,8-pyrenoquinodimethanes fused by indene units, respectively, were synthesized by reaction of 1,4,5,8-tetrakis(mesitylethynyl)naphthalene (7) with bis(2,4,6-trimethylpyridine)iodine(I) hexafluorophosphate via cationic cyclization mechanisms at both centers of reaction. Unexpectedly, reaction of 7 with iodine, a reagent that typically gives products of cationic cyclization, gave cycloheptapentalenoindenophenalene derivative 9b and its diastereomer incorporating an azulene unit at one end of the π framework, via two different modes (radical and cationic) of cyclization at each reaction site. The physical properties of the products are presented, and the dual modes of cyclization of 7 and its model compound with only one reaction center are discussed.

Embryonic tongue morphogenesis in an organ culture model of mouse mandibular arches: blocking Sonic hedgehog signaling leads to microglossia.

Mouse tongue development is initiated with the formation of lateral lingual swellings just before fusion between the mediodorsal surfaces of the mandibular arches at around embryonic day 11.0. Here, we investigated the role of Sonic hedgehog (Shh) signaling in embryonic mouse tongue morphogenesis. For this, we used an organ culture model of the mandibular arches from mouse embryos at embryonic day 10.5. When the Shh signaling inhibitor jervine was added to the culture medium for 24-96 h, the formation of lateral lingual swellings and subsequent epithelial invagination into the mesenchyme were impaired markedly, leading to a hypoplastic tongue with an incomplete oral sulcus. Notably, jervine treatment reduced the proliferation of non-myogenic mesenchymal cells at the onset of forming the lateral lingual swellings, whereas it did not affect the proliferation and differentiation of a myogenic cell lineage, which created a cell community at the central circumferential region of the lateral lingual swellings as seen in vivo and in control cultures lacking the inhibitor. Thus, epithelium-derived Shh signaling stimulates the proliferation of non-myogenic mesenchymal cells essential for forming lateral lingual swellings and contributes to epithelial invagination into the mesenchyme during early tongue development.

Improvement of fluctuations of intraocular pressure after cataract surgery in primary angle closure glaucoma patients.

BACKGROUND: Cataract surgery has been shown to reduce intraocular pressure (IOP) in eyes with primary angle closure glaucoma (PACG). In this study, we examined IOP fluctuations and circadian IOP patterns before and after cataract surgery in PACG patients using the contact lens sensor (CLS) Triggerfish. METHODS: This was a prospective open-label study. Ten consecutive patients with PACG were included. The patients underwent phacoemulsification and intraocular lens implantation (PEA+IOL) surgery. In each eye, IOP fluctuations over 24 h were measured with the CLS before and at three months after the surgery. Changes in corneal curvatures and anatomic parameters of the anterior chamber were measured by anterior segment optical coherence tomography (AS-OCT). RESULTS: The mean IOP was significantly reduced from 14.7 ± 1.5 mmHg to 11.2 ± 2.2 mmHg at three months after the surgery in PACG patients (P = 0.002). Although the mean range of 24-h IOP fluctuations was not significantly changed after the surgery (P = 0.49), the mean range of IOP fluctuations during the nocturnal period was significantly decreased from 246 ± 61 mVeq to 179 ± 64 mVeq after the surgery (P = 0.02). After the surgery, seven eyes of the ten (70%) showed the same circadian IOP patterns. AS-OCT showed significant improvements in the anatomic parameters of the anterior chamber after the surgery. CONCLUSIONS: Cataract surgery decreased IOP fluctuations during the nocturnal period in PACG patients. These effects might partly prevent the progression of PACG.

Molecular contribution to cleft palate production in cleft lip mice.

Cleft palate following cleft lip may include a developmental disorder during palatogenesis. CL/Fr mice fetuses, which develop cleft lip and palate spontaneously, have less capability for in vivo cell proliferation in palatal mesenchyme compared with CL/Fr normal fetuses. In order to know the changes of signaling molecules contributing to cleft palate morphogenesis following cleft lip, the mRNA expression profiles were compared in palatal shelves oriented vertically (before elevation) in CL/Fr fetuses with or without cleft lip. The changes in mRNA profile of cleft palate morphogenesis were presented in a microarray analysis, and genes were restricted to lists contributing to cleft palate development in CL/Fr fetuses with cleft lip. Four candidate genes (Ywhab, Nek2, Tacc1 and Frk) were linked in a gene network that associates with cell proliferation (cell cycle, MAPK, Wnt and Tgf beta pathways). Quantitative real-time RT-PCR highlighted the candidate genes that significantly changed in CL/Fr fetuses with cleft lip (Ywhab, Nek2 and Tacc1). The results of these molecular contributions will provide useful information for a better understanding of palatogenesis in cleft palate following cleft lip. Our data indicated the genetic contribution to cleft palate morphogenesis following cleft lip.

Generation of a mouse model with down-regulated U50 snoRNA (SNORD50) expression and its organ-specific phenotypic modulation.

Box C/D-type small nucleolar RNAs (snoRNAs) are functional RNAs responsible for mediating 2'-O-ribose methylation of ribosomal RNAs (rRNAs) within the nucleolus. In the past years, evidence for the involvement of human U50 snoRNA in tumorigenesis has been accumulating. We previously identified U50HG, a non-protein-coding gene that hosted a box C/D-type U50 snoRNA, in a chromosomal breakpoint in a human B-cell lymphoma. Mouse genome analysis revealed four mouse U50 (mU50) host-genes: three mU50HG-a gene variants that were clustered in the genome and an mU50HG-b gene that we supposed to be the U50HG ortholog. In this study, to investigate the physiological importance of mU50 snoRNA and its involvement in tumorigenesis, we eliminated mU50 snoRNA sequences from the mU50HG-b gene. The established mouse line (ΔmU50(HG-b)) showed a significant reduction of mU50 snoRNA expression without alteration of the host-gene length and exon-intron structure, and the corresponding target rRNA methylation in various organs was reduced. Lifelong phenotypic monitoring showed that the ΔmU50(HG-b) mice looked almost normal without accelerated tumorigenicity; however, a notable difference was the propensity for anomalies in the lymphoid organs. Transcriptome analysis showed that dozens of genes, including heat shock proteins, were differentially expressed in ΔmU50(HG-b) mouse lymphocytes. This unique model of a single snoRNA knockdown with intact host-gene expression revealed further new insights into the discrete transcriptional regulation of multiple mU50 host-genes and the complicated dynamics involved in organ-specific processing and maintenance of snoRNAs.

Molecular signaling at the fusion stage of the mouse mandibular arch: involvement of insulin-like growth factor family.

Fusion of the branchial arch derivatives is a crucial event in the development of the craniofacial architecture. Here, we surveyed the gene expression profile, focusing on the fusion process of the mouse mandibular arch at embryonic day 10.5. In order to identify the genes that are relevant to the midline fusion process, we subdivided the mandibular arch medially and laterally, and determined gene expression using microarray and real-time quantitative PCR. By comparing the transcriptomes of the medial and lateral regions, 362 genes were identified as medial region-specific genes, while 346 genes were designated lateral region-specific. Taken with Gene Ontology analysis, KEGG pathways and Ingenuity Pathway Analysis (IPA), a survey of the medial region-specific gene dataset revealed significant expression of the insulin-like growth factor (Igf) family as well as other growth factor families (Hh, Wnt, Tgf-Bmp, Mapk-Fgf and Notch). To determine the discrete expression pattern of Igf family genes in the medial region, we microdissected the medial part of the mandibular arch into epithelial and mesenchymal components, and found that Igf1 was highly expressed in the mesenchyme, Igf2 and Igf1r were expressed in both the midline epithelium and surrounding mesenchyme, and Igfbp5 was highly expressed in the epithelium. Immunohistochemical findings validated the regional Igf gene expression profiles. Our observations suggest that in the “merging” fusion of the mandibular arch, the Igf cascade may contribute to generation of proliferation pressure from the mesenchyme and preservation of epithelial phenotypes and architecture during mesenchymal confluence.

[Severe hypotension and ventricular fibrillation during combined general and epidural anesthesia in a patient on major tranquilizers].

A 54-year-old man (height 155 cm, weight 49 kg) was scheduled for retroperitoneoscopic nephrectomy. He had a history of schizophrenia that had been controlled with propericiazine 10 mg and bromperidol 3 mg daily for 34 years. After induction of anesthesia, 1% mepivacaine 5 ml was administered via an epidural catheter. Blood pressure decreased 15 minutes later to 47/25 mmHg and heart rate dropped to 50 beats x min(-1). Ventricular fibrillation occurred despite titrated injection of ephedrine (40 mg total), phenylephrine (1 mg total), atropine (0.5 mg total), and rapid infusion of crystalloid and colloid solutions. Chest compression and defibrillation were required to restore spontaneous circulation. Surgery was cancelled and he was extubated 45 minutes later without any complications. These findings suggest that caution must be exercised when combining general and epidural anesthesia for patients on long-term major tranquilizers. In the event of refractory hypotension, the use of direct-acting vasoconstrictors such as noradrenaline or vasopressin should be considered.

Treatment of macular edema due to branch retinal vein occlusion with single or multiple intravitreal injections of bevacizumab.

PURPOSE: We examined the predictive factors for final visual acuity (VA) with macular edema of branch retinal vein occlusion (BRVO) treated by intravitreal injection of bevacizumab (IVB) and examined the differences between patients without recurrent macular edema due to BRVO after a single IVB and patients treated with multiple IVB because of recurrent macular edema. METHODS: In this retrospective study, 37 eyes of 37 patients with BRVO were treated with IVB and followed up for more than 24 weeks. Eighteen eyes showed no recurrence of macular edema after a single IVB (single IVB group). The remaining 19 eyes showed recurrent macular edema and underwent multiple IVB (multiple IVB group). VA and morphologic parameters of optical coherence tomography were examined. RESULTS: Mean VA, central retinal thickness, and mean retinal thickness in a circular region of 1-mm diameter at the fovea improved significantly with IVB treatment in both groups. Final VA was correlated with baseline VA and integrity grade of the photoreceptor inner and outer segment (IS/OS) line beneath the fovea. CONCLUSION: Baseline VA and IS/OS line grade at 4 weeks may be predictive factors for final VA.

One-year results of photodynamic therapy combined with intravitreal ranibizumab for exudative age-related macular degeneration.

Purpose. To evaluate the effects of photodynamic therapy (PDT) combined with intravitreal injection of ranibizumab (IVR) for exudative age-related macular degeneration (AMD). Methods. Retrospective case series. Thirty eight eyes of 38 patients with exudative AMD underwent combined therapy consisting first of IVR, followed by PDT within a week and the second IVR at 1 month. All patients were followed up for more than 12 months. The best corrected visual acuity (BCVA) and central macular thickness (CMT) were examined. Results. The mean number of IVR and PDT sessions were 2.9 ± 1.3 and 1.1 ± 0.3, respectively. The mean BCVA and CMT were significantly improved to 0.38 logMAR units (P < 0.01) and 240 µm (P < 0.01) at 12 months, respectively. Thirty-six of 38 eyes (94.8%) improved or maintained BCVA at 12 months. Conclusion. PDT combined with IVR for exudative AMD was effective at improving visual acuity and CMT with a low recurrence rate for 12 months.

Intraoperative changes in idiopathic macular holes by spectral-domain optical coherence tomography.

PURPOSE: To examine anatomical changes in idiopathic macular holes during surgery using handheld spectral-domain optical coherence tomography (SD-OCT). METHODS: Five eyes of 5 patients who underwent surgery for the repair of idiopathic macular holes were examined. The surgery included standard 25-gauge, 3-port pars plana vitrectomy, removal of the internal limiting membrane (ILM), fluid-air exchange, and 20% sulfur hexafluoride tamponade. Intraoperative SD-OCT images of the macular holes were obtained after ILM removal and under fluid-air exchange using a handheld SD-OCT. From SD-OCT images, the macular hole base diameter (MHBD) was measured and compared. RESULTS: All macular holes were successfully closed after the primary surgery. The mean MHBD under fluid-air exchange was significantly smaller than the mean MHBD after ILM removal and the preoperative mean MHBD. In 1 eye with a stage 3 macular hole, SD-OCT images revealed that the inner edges of the macular hole touched each other under fluid-air exchange. CONCLUSION: Fluid-air exchange significantly reduced MHBD during surgery to repair macular holes. Fluid-air exchange may be an important step for macular hole closure as it reduces the base diameter of the macular hole.

Short-term results of canaloplasty surgery for primary open-angle glaucoma in Japanese patients.

PURPOSE: To report surgical results of canaloplasty surgery for primary open-angle glaucoma (POAG) in Japanese patients. METHODS: Eleven eyes of 9 POAG patients underwent canalopasty surgery at Toyama University Hospital. Three eyes of 3 patients underwent canaloplasty alone and 8 eyes of 6 patients underwent canaloplasty combined with cataract surgery. Canaloplasty was performed with a 10-0 polypropylene tensioning suture and an iTrack™ 250A microcatheter. All patients were followed up for 12 months. Changes in intraocular pressure (IOP) and postoperative complications were examined. RESULTS: Mean preoperative IOP was 23.4 ± 5.5 mm Hg. Mean number of antiglaucoma drops was 2.8 ± 0.6 before canaloplasty and decreased to 1.2 ± 0.8 at 12 months after canaloplasty (p < 0.01). Mean IOP decreased postoperatively, being 13.7 ± 2.8 mm Hg at 1 month, 12.8 ± 3.5 mm Hg at 3 months, 14.0 ± 4.4 mm Hg at 6 months, and 15.0 ± 4.1 mm Hg at 12 months. The most frequent postoperative complication was mild hyphema (45.5%), which disappeared within 14 days after surgery. CONCLUSIONS: Canaloplasty may be an alternative surgery for POAG patients to reduce IOP to a value of approximately 15 mm Hg.

A depressurization assistance control based on the posture of a seated patient on a wheelchair.

For reducing the risk of pressure sore caused by long period sitting on a wheelchair, we develop a depressurization motion assistance system which is low cost and suitable for practical use. Our developing system consists of a seating cushion which the patient sits on and four air cells which can lift or incline the seating cushion. Each air cell is actuated by small air compressor, which can drive using batteries on the wheelchair respectively, and each compressor has a pressure sensor on its body. In this paper, our key ideas are two topics. One topic is mechanical design for practical use. We realize thin mechanism which enables easy implementation to the general wheelchair. For realizing this thinly design, we develop the tilt mechanism using elasticity of acrylic resin and the controller which uses only pressure sensors for estimating its lifting height and inclination. The other topic is assistance control scheme based on the patient's depressurization operation for increasing a rehabilitation performance. For realizing the proposed control scheme, we analyze the hip depressurization operation by the nursing specialists and use its results for estimating the patient's condition. Using our system, the patient can depressurize by his own will on the general wheelchair easily. The performance of our system is verified by experiments using our prototype.