Identification and Characterization of Proteins That Are Involved in RTP1S-Dependent Transport of Olfactory Receptors.

Olfaction is mediated via olfactory receptors (ORs) that are expressed on the cilia membrane of olfactory sensory neurons in the olfactory epithelium. The functional expression of most ORs requires the assistance of receptor-transporting proteins (RTPs). We examined the interactome of RTP1S and OR via proximity biotinylation. Deubiquitinating protein VCIP135, the F-actin-capping protein sub-unit alpha-2, and insulin-like growth factor 2 mRNA-binding protein 2 were biotinylated via AirID fused with OR, RTP1S-AirID biotinylated heat shock protein A6 (HSPA6), and double-stranded RNA-binding protein Staufen homolog 2 (STAU2). Co-expression of HSPA6 partially enhanced the surface expression of Olfr544. The surface expression of Olfr544 increased by 50-80%. This effect was also observed when RTP1S was co-expressed. Almost identical results were obtained from the co-expression of STAU2. The interactions of HSPA6 and STAU2 with RTP1S were examined using a NanoBit assay. The results show that the RTP1S N-terminus interacted with the C-terminal domain of HSP6A and the N-terminal domain of STAU2. In contrast, OR did not significantly interact with STAU2 and HSPA6. Thus, HSP6A and STAU2 appear to be involved in the process of OR traffic through interaction with RTP1S.

Endoscopic treatment of Bouveret syndrome with a combination of electrohydraulic lithotripsy and balloon expansion: A case report.

Bouveret syndrome is a rare type of ileus caused by the impaction of gallstones passing through a cholecystoenteric fistula in the duodenum. Endoscopic treatment with minimally invasive procedures is preferable for patients with this syndrome, typically for elderly individuals with a high surgical risk. Conventional endoscopic techniques often fail to remove impacted stones that are generally large and occasionally solid. We report the case of an 88-year-old bedridden woman with severe dementia who presented with difficulty in breathing. The patient was diagnosed with aspiration pneumonia. In addition, computed tomography showed a cholecystoduodenal fistula and a gallstone 37 mm in diameter that impacted the duodenal bulb. Bouveret syndrome was diagnosed on the basis of the computed tomography findings. The impacted stone was too large and hard to split with standard endoscopic lithotripsy using grasping forceps, mechanical lithotripter, polypectomy snare, basket catheter, and electrohydraulic lithotripsy (EHL). However, EHL with a dual-channel therapeutic endoscope was achieved to drill a narrow hole approximately 20 mm deep into the stone, in four sessions. The stone was subsequently split by inflating the balloon, which was inserted into the hole, to 10 mm in diameter at 3 atm. All the split stones were spontaneously excreted during defecation after a few days. If the gallstone is too hard to fragment by endoscopic EHL alone, a combination of EHL and balloon expansion might be a useful alternative.

Mirizzi Syndrome Type IV Successfully Treated with Peroral Single-operator Cholangioscopy-guided Electrohydraulic Lithotripsy: A Case Report with Literature Review.

A 76-year-old man presented with liver dysfunction and intrahepatic bile duct dilatation. Imaging studies showed two large stones that had become impacted in the common hepatic duct, which was fused with the gallbladder. The patient was diagnosed with Mirizzi syndrome type IV. Hepaticojejunostomy and stone removal failed due to dense gallbladder adhesions involving the right hepatic artery. The bile flow was temporarily restored; however, the patient experienced cholangitis 16 months later. The stones were extracted via peroral single-operator cholangioscopy (SOC)-guided electrohydraulic lithotripsy. This is the first case in which stones were completely removed by SOC-guided treatment in a patient with Mirizzi syndrome type IV.

Modulation of serotonin in the gut-liver neural axis ameliorates the fatty and fibrotic changes in non-alcoholic fatty liver.

The etiology of non-alcoholic fatty liver disease (NAFLD) consists of various factors, including neural signal pathways. However, the molecular mechanisms of the autonomic neural signals influencing NAFLD progression have not been elucidated. Therefore, we examined the involvement of the gut-liver neural axis in NAFLD development and tested the therapeutic effect of modulation of this axis in this study. To test the contribution of the gut-liver neural axis, we examined NAFLD progression with respect to body weight, hepatic steatosis, fibrosis, intestinal tight junction, microbiota and short-chain fatty acids in NAFLD models of choline-deficient defined L-amino-acid and high-fat diet-fed mice with or without blockades of autonomic nerves from the liver. Blockade of the neural signal from the liver to the gut in these NAFLD mice models ameliorated the progression of liver weight, hepatic steatosis and fibrosis by modulating serotonin expression in the small intestine. It was related to the severity of the liver pathology, the tight junction protein expression, microbiota diversity and short-chain fatty acids. These effects were reproduced by administrating serotonin antagonist, which ameliorated the NAFLD progression in the NAFLD mice models. Our study demonstrated that the gut-liver neural axis is involved in the etiologies of NAFLD progression and that serotonin expression through this signaling network is the key factor of this axis. Therefore, modulation of the gut-liver neural axis and serotonin antagonist ameliorates fatty and fibrotic changes in non-alcoholic fatty liver, and can be a potential therapeutic target of NAFLD.This article has an associated First Person interview with the first author of the paper.

A Cross-Sectional Study of Age-Related Changes in Oral Function in Healthy Japanese Individuals.

BACKGROUND: Oral function deterioration is related to a variety of factors, including aging, decline in activities of daily living, malnutrition, and cognitive decline. This cross-sectional study examined the effects of aging on oral function in healthy individuals. METHODS: A retrospective study was conducted on 175 healthy, independent patients aged 40-89 years, without dementia and with ≥20 teeth, who visited a local dental clinic in Japan. Patients were compared with 92 university students aged 20-29 years. The seven criteria proposed by the Japanese Society of Gerodontology to diagnose "oral hypofunction" were observed and statistically analyzed. RESULTS: Compared with those in the control group, the degree of tongue coating was increased in the group aged over 80 years, occlusal force was decreased in the group aged 70-79 years, tongue motor function was decreased in the groups aged 60-69 years and older, and tongue pressure was decreased in the groups aged 70-79 years and older. CONCLUSIONS: Healthy, independent individuals maintained several oral function criteria across aging, including oral mucosal wetness, occlusal force, lip motor function, masticatory function, and swallowing function. Tongue motor function and tongue pressure decreased with aging, indicating that these may be rehabilitation targets.

Ghrelin-insulin-like growth factor-1 axis is activated via autonomic neural circuits in the non-alcoholic fatty liver disease.

BACKGROUND: The correlation of the growth hormone (GH) and insulin-like growth factor-1 (IGF-1) with non-alcoholic fatty liver disease (NAFLD) has been reported in epidemiological studies. However, the mechanisms of molecular and inter-organ systems that render these factors to influence on NAFLD have not been elucidated. In this study, we examined the induction of ghrelin which is the GH-releasing hormone and IGF-1, and involvement of autonomic neural circuits, in the pathogenesis of NAFLD. METHODS: The expression of gastric and hypothalamic ghrelin, neural activation in the brain, and serum IGF-1 were examined in NAFLD models of choline-deficient defined l-amino-acid diet-fed, melanocortin 4 receptor knockout mice, and partial hepatectomy mice with or without the blockades of autonomic nerves to test the contribution of neural circuits connecting the brain, liver, and stomach. KEY RESULTS: The fatty changes in the liver increased the expression of gastric ghrelin through the autonomic pathways which sends the neural signals to the arcuate nucleus in the hypothalamus through the afferent vagal nerve which reached the pituitary gland to release GH and then stimulate the IGF-1 release from the liver. In addition, high levels of ghrelin expression in the arcuate nucleus were correlated with NAFLD progression regardless of the circuits. CONCLUSIONS: Our study demonstrated that the fatty liver stimulates the autonomic nervous signal circuits which suppress the progression of the disease by activating the gastric ghrelin expression, the neural signal transduction in the brain, and the release of IGF-1 from the liver.

The N-terminal region of RTP1S plays important roles in dimer formation and odorant receptor-trafficking.

Receptor-transporting protein 1S (RTP1S) is an accessory protein that mediates the transport of mammalian odorant receptors (ORs) into the plasma membrane. Although most ORs fail to localize to the cell surface when expressed alone in nonolfactory cells, functional expression of ORs is achieved with the coexpression of RTP1S. However, the mechanism for RTP1S-mediated OR trafficking remains unclear. In this study, we attempted to reveal the mode of action and critical residues of RTP1S in OR trafficking. Experiments using N-terminal truncation and Ala substitution mutants of RTP1S demonstrated that four N-terminal amino acids have essential roles in OR trafficking. Additionally, using recombinant proteins and split luciferase assays in mammalian cells, we provided evidence for the dimer formation of RTP1S. Furthermore, we determined that the 2nd Cys residue is required for the efficient dimerization of RTP1S. Altogether, these findings provide insights into the mechanism for plasma membrane transport of ORs by RTP1S.

Inhibition of sodium glucose cotransporter 2 (SGLT2) delays liver fibrosis in a medaka model of nonalcoholic steatohepatitis (NASH).

The rise in the incidence of nonalcoholic steatohepatitis (NASH) has necessitated the development of an effective prevention methodology. An antidiabetic drug, belonging to the group of sodium glucose cotransporter 2 (SGLT2) inhibitors, has been tested for its therapeutic effect on NASH; however, no studies to date have demonstrated the preventive effect of an SGLT2 inhibitor on the histological progression of steatosis and fibrosis in a sequential manner in animal models. In the present study, we examined the effect of the SGLT2 inhibitor, tofogliflozin (Tofo), on NASH liver tissue using medaka as an animal model, maintaining a feeding amount and drug concentration in all animal bodies. We generated a medaka NASH model by feeding d-rR/Tokyo medaka a high-fat diet and administered Tofo by dissolving the drug directly in the water of the feeding tank. Thereafter, the effects of Tofo on body weight (BW), liver weight, hepatotoxicity, fatty infiltration, and fibrotic changes in the liver were examined. We report here that SGLT2 is expressed in medaka fish and that Tofo inhibits the accumulation of fatty tissue and delays the progression of liver fibrosis in the medaka NASH model by inhibiting increases in blood sugar, serum lipids, and transaminase, irrespective of changes in BW. These results suggest that Tofo is effective for treating NASH and that the medaka model may be useful for developing new therapeutic drugs for this disease.

Autonomic nervous system network and liver regeneration.

To date, various signal transducers, cytokines, growth factors, and hormones have been reported to play an important role in homeostasis of various organs. Various cells and organs are involved in the hepatic regeneration process, which proceeds as a result of the coordination of many factors. While these factors are well known to be involved in the liver regeneration after the liver injury, however, as the details of such mechanisms have not been sufficiently elucidated, the practical applicability of hepatic regeneration based on the action of these and cytokines growth factors is still unclear. In terms of the involvement of the autonomic nervous system in hepatic regeneration, cell proliferation resulting from direct signal transduction to the liver has also been reported and recent studies focusing on the inter-organ communication via neural network opened a novel aspect of this field for therapeutic applicability. Therefore, the appropriate understanding of the relationship between autonomic neural network and liver regeneration through various organs including brain, afferent nerve, efferent nerve, etc. is essential. This mini-review explains the principle of neural system involved in the inter-organ communication and its contribution on the liver regeneration upon the liver injury reviewing recent progress in this field.

Effect of a neural relay on liver regeneration in mice: activation of serotonin release from the gastrointestinal tract.

The development of therapeutic options to promote hepatic regeneration following severe liver injury is essential. While humoral factors have been reported as mechanisms of liver regeneration, the contributions of interorgan communication to liver regeneration have not been reported. In this study, we examined the effect of a neural relay on liver regeneration via activation of serotonin release from the gastrointestinal (GI) tract. Our results demonstrated that the afferent visceral nerve from the liver activates the efferent vagus nerve from the brain, leading to activation of serotonin release from the GI tract and contributing to liver regeneration. While it is difficult to apply these results directly to human health, we believe that this study may represent a step toward developing essential therapeutics to promote liver regeneration.

Effect of histidine on sorafenib-induced vascular damage: Analysis using novel medaka fish model.

BACKGROUND: Sorafenib (SFN) is an anti-angiogenic chemotherapeutic that prolongs survival of patients with hepatocellular carcinoma (HCC); its side effects, including vascular damages such as hand-foot syndrome (HFS), are a major cause of therapy discontinuation. We previously reported that maintenance of peripheral blood flow by intake of dried bonito broth (DBB) significantly prevented HFS and prolonged the administration period. The amino acids contained in DBB probably contribute to its effects, but the mechanism has not been clarified. We hypothesized that histidine, the largest component among the amino acids contained in DBB, has effects on SFN-induced vascular damage, and evaluated this possibility using a novel medaka fish model. METHODS: The fli::GFP transgenic medaka fish model has a fluorescently visible systemic vasculature. We fed the fish with SFN with and without histidine to compare blood flow and vascular structure among the differently fed models. The vascular cross-sectional area of each fish was measured to determine vascular diameter changes. RESULTS: Our results demonstrated that SFN-fed medaka developed a narrower vascular diameter. In addition, this narrowing was counteracted by addition of histidine to the medaka diet. We observed no positive effect of histidine on regeneration of cut vessels or on cell growth of endothelial cells and HCC cell lines. CONCLUSION: We proved the efficacy of the medaka model to assess vascular changes after administration of specific chemicals. And our results suggest that SFN causes vascular damage by narrowing peripheral vessel diameter, and that histidine effectively counteracts these changes to maintain blood flow.

Successful Fitting of a Complete Maxillary Denture in a Patient with Severe Alzheimer's Disease Complicated by Oral Dyskinesia.

There is an increasing population of elderly patients suffering from Alzheimer's disease (AD), the most common form of dementia. In dentistry, a critical problem associated with these patients is the use of a new denture, as AD patients often refuse dental management and are disturbed by minor changes in their oral environment. Some AD patients have further complications associated with oral dyskinesia, a movement disorder that can make dental management difficult, including the stability of a complete denture. In this case, we successfully fitted a complete maxillary denture using modified bilateral balanced occlusion after multiple tooth extractions under intravenous sedation in a 66-year-old woman with severe AD complicated by oral dyskinesia. Following treatment, her appetite and food intake greatly improved. Providing a well-fitting complete denture applied by modified bilateral balanced occlusion, which removes lateral interference using zero-degree artificial teeth for movement disorder of the jaw in patients with severe AD complicated by oral dyskinesia, helps improve oral function.

Effects of Fibrotic Tissue on Liver-targeted Hydrodynamic Gene Delivery.

Hydrodynamic gene delivery is a common method for gene transfer to the liver of small animals, and its clinical applicability in large animals has been demonstrated. Previous studies focused on functional analyses of therapeutic genes in animals with normal livers and little, however, is known regarding its effectiveness and safety in animals with liver fibrosis. Therefore, this study aimed to examine the effects of liver fibrosis on hydrodynamic gene delivery efficiency using a rat liver fibrosis model. We demonstrated for the first time, using pCMV-Luc plasmid, that this procedure is safe and that the amount of fibrotic tissue in the liver decreases gene delivery efficiency, resulting in decrease in luciferase activity depending on the volume of fibrotic tissue in the liver and the number of hepatocytes that are immunohistochemically stained positive for transgene product. We further demonstrate that antifibrotic gene therapy with matrix metalloproteinase-13 gene reduces liver fibrosis and improves efficiency of hydrodynamic gene delivery. These results demonstrate the negative effects of fibrotic tissue on hydrodynamic gene delivery and its recovery by appropriate antifibrotic therapy.

Speaking Tracheostomy Tube and Modified Mouthstick Stylus in a Ventilator-Dependent Patient with Spinal Cord Injury.

Communication is a serious problem for patients with ventilator-dependent tetraplegia. A 73-year-old man was presented at the emergency room in cardiopulmonary arrest after falling from a height of 2 m. After successful resuscitation, fractures of the cervical spine and cervical spinal cord injury were found. Due to paralysis of the respiratory muscles, a mechanical ventilator with a tracheostomy tube was required. First, a cuffed tracheostomy tube and a speaking tracheostomy tube were inserted, and humidified oxygen was introduced via the suction line. Using these tubes, the patient could produce speech sounds, but use was limited to 10 min due to discomfort. Second, a mouthstick stylus, fixed on a mouthpiece that fits over the maxillary teeth, was used. The patient used both a communication board and a touch screen device with this mouthstick stylus. The speaking tracheostomy tube and mouthstick stylus greatly improved his ability to communicate.

Selective Formation of Zigzag Edges in Graphene Cracks.

We report the thermally induced unconventional cracking of graphene to generate zigzag edges. This crystallography-selective cracking was observed for as-grown graphene films immediately following the cooling process subsequent to chemical vapor deposition (CVD) on Cu foil. Results from Raman spectroscopy show that the crack-derived edges have smoother zigzag edges than the chemically formed grain edges of CVD graphene. Using these cracks as nanogaps, we were also able to demonstrate the carrier tuning of graphene through the electric field effect. Statistical analysis of visual observations indicated that the crack formation results from uniaxial tension imparted by the Cu substrates together with the stress concentration at notches in the polycrystalline graphene films. On the basis of simulation results using a simplified thermal shrinkage model, we propose that the cooling-induced tension is derived from the transient lattice expansion of narrow Cu grains imparted by the thermal shrinkage of adjacent Cu grains.

Image-Guided Hydrodynamic Gene Delivery: Current Status and Future Directions.

Hydrodynamics-based delivery has been used as an experimental tool to express transgene in small animals. This in vivo gene transfer method is useful for functional analysis of genetic elements, therapeutic effect of oligonucleotides, and cancer cells to establish the metastatic cancer animal model for experimental research. Recent progress in the development of image-guided procedure for hydrodynamics-based gene delivery in large animals directly supports the clinical applicability of this technique. This review summarizes the current status and recent progress in the development of hydrodynamics-based gene delivery and discusses the future directions for its clinical application.

Dual signaling pathways of arterial constriction by extracellular uridine 5'-triphosphate in the rat.

We investigated actions of uridine 5'-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 µM) elicited an inward-rectifying current strongly reminiscent of activation of P2X(1) receptor, and a similar current was also induced by α,ß-methylene adenosine 5'-triphosphate (ATP) (≥100 nM). UTP desensitized α,ß-methylene ATP-evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X(1)-receptor antibody. Both UTP (1 mM) and α,ß-methylene ATP (10 µM) elicited similar conductance single channel activities. UTP (≥10 µM) provoked a dose-dependent contraction of de-endothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca(2+) or bath-applied 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 µM) or nifedipine (10 µM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 µM). Similar biphasic rises in [Ca(2+)](i) were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X(1) receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X(1)-like and P2Y receptors, but not TRPC3.

Binding specificity of the recombinant cytoplasmic domain of Cordyceps militaris ß-1,3-glucan synthase catalytic subunit.

The cytoplasmic domain of the medicinal mushroom Cordyceps militaris ß-1,3-glucan synthase catalytic subunit Fks1 was expressed as a fusion protein with an N-terminal hexahistidine tag and glutathione S-transferase in an Escherichia coli cell-free translation system, and was assayed for binding specificity. The recombinant cytoplasmic domain bound specifically to UDP-agarose and lichenan (ß-glucan), but not to ADP-agarose, GDP-agarose, or other carbohydrates.

Vitamin K-deficient intracranial hemorrhage as the first symptom of cytomegalovirus hepatitis with cholestasis.

Since vitamin K2 (VitK2) syrup prophylaxis has become a routine measure for neonates and young infants, the incidence of vitamin K deficiency (VitK-D) in infancy has markedly decreased. However, we recently experienced 2 infantile cases of VitK deficiency, in whom intracranial hemorrhage (ICH) was the first clinical sign of CMV hepatitis. Case 1 is a breast-fed boy who received VitK2 syrup orally at birth and at the age of 1 month. He did not suckle well and developed a generalized tonic convulsion twice at the age of 8 weeks. Case 2 is a mixed-fed boy who also received VitK2 syrup twice but developed vomiting and drowsiness at the age of 4 months. In both cases, laboratory tests showed anemia, leukocytosis, liver dysfunction with cholestasis, and coagulopathy, consistent with VitK-D abnormality. Their serological analyses showed that cytomegalovirus (CMV) IgG and IgM were both positive. In case 1, CMV DNA was positive, as judged by the PCR method. In case 2, CMV antigenemia was positive. Hence we diagnosed these two patients as having VitK-D ICH caused by CMV hepatitis with cholestasis. CMV hepatitis is a risk factor of VitK-D ICH.