👤 Hiroyuki Saito

Large-scale genome-wide association studies (GWASs) on bipolar disorder (BD) have implicated the involvement of the fatty acid desaturase (FADS) locus. These enzymes (FADS1 and FADS2) are involved in the metabolism of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are thought to potentially benefit patients with mood disorders. To model reductions in the activity of FADS1/2 affected by the susceptibility alleles, we generated mutant mice heterozygously lacking both Fads1/2 genes. We measured wheel-running activity over six months and observed bipolar swings in activity, including hyperactivity and hypoactivity. The hyperactivity episodes, in which activity was far above the norm, usually lasted half a day; mice manifested significantly shorter immobility times on the behavioral despair test performed during these episodes. The hypoactivity episodes, which lasted for several weeks, were accompanied by abnormal circadian rhythms and a marked decrease in wheel running, a spontaneous behavior associated with motivation and reward systems. We comprehensively examined lipid composition in the brain and found that levels of certain lipids were significantly altered between wild-type and the heterozygous mutant mice, but no changes were consistent with both sexes and either DHA or EPA was not altered. However, supplementation with DHA or a mixture of DHA and EPA prevented these episodic behavioral changes. Here we propose that heterozygous Fads1/2 knockout mice are a model of BD with robust constitutive, face, and predictive validity, as administration of the mood stabilizer lithium was also effective. This GWAS-based model helps to clarify how lipids and their metabolisms are involved in the pathogenesis and treatment of BD. Show less

Central neurocytoma (CN) is classically defined by its intraventricular location, neuronal/neurocytic differentiation, and histological resemblance to oligodendroglioma. Extraventricular neurocytoma (EVN) shares similar histological features with CN, while it distributes any site without contact with the ventricular system. CN and EVN have distinct methylation landscapes, and EVN has a signature fusion gene, FGFR1-TACC1. These characteristics distinguish between CN and EVN. A 30-year-old female underwent craniotomy and resection of a left intraventricular tumor at our institution. The histopathology demonstrated the classical findings of CN. Adjuvant irradiation with 60 Gy followed. No recurrence has been recorded for 25 years postoperatively. RNA sequencing revealed FGFR1-TACC1 fusion and methylation profile was discrepant with CN but compatible with EVN. We experienced a case of anatomically and histologically proven CN in the lateral ventricle. However, the FGFR1-TACC1 fusion gene and methylation profiling suggested the molecular diagnosis of EVN. The representative case was an "intraventricular" neurocytoma displaying molecular features of an "extraventricular" neurocytoma. Clinicopathological and molecular definitions have collided in our case and raised questions about the current definition of CN and EVN. Show less

Hypertrophic cardiomyopathy (HCM) is the most common heart disease in cats with a suspected genetic origin. Previous studies have identified five HCM-associated variants in three genes (Myosin binding protein C3: MYBPC3 p.A31P, p.A74T, p.R820W; Myosin heavy chain 7: MYH7 p.E1883K; Alstrom syndrome protein 1: ALMS1 p.G3376R). These variants are considered breed-specific, with the exception of MYBPC3 p.A74T, and have rarely been found in other breeds. However, genetic studies on HCM-associated variants across breeds are still insufficient because of population and breed bias caused by differences in genetic background. This study investigates the ubiquitous occurrence of HCM-associated genetic variants among cat breeds, using 57 HCM-affected, 19 HCM-unaffected, and 227 non-examined cats from the Japanese population. Genotyping of the five variants revealed the presence of MYBPC3 p.A31P and ALMS1 p.G3376R in two (Munchkin and Scottish Fold) and five non-specific breeds (American Shorthair, Exotic Shorthair, Minuet, Munchkin and Scottish Fold), respectively, in which the variants had not been identified previously. In addition, our results indicate that the ALMS1 variants identified in the Sphynx breed might not be Sphynx-specific. Overall, our results suggest that these two specific variants may still be found in other cat breeds and should be examined in detail in a population-driven manner. Furthermore, applying genetic testing to Munchkin and Scottish Fold, the breeds with both MYBPC3 and ALMS1 variants, will help prevent the development of new HCM-affected cat colonies. Show less

The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVN Using a combination of viral tools, we mapped PVN We found that PVN Our results elucidate the neuroanatomical organization of PVN Show less

The melanin-concentrating hormone (MCH) system is involved in numerous functions, including energy homeostasis, food intake, sleep, stress, mood, aggression, reward, maternal behavior, social behavior, and cognition. In rodents, MCH acts on MCHR1, a G protein-coupled receptor, which is widely expressed in the brain and abundantly localized to neuronal primary cilia. Cilia act as cells' antennas and play crucial roles in cell signaling to detect and transduce external stimuli to regulate cell differentiation and migration. Cilia are highly dynamic in terms of their length and morphology; however, it is not known if cilia length is causally regulated by MCH system activation in vivo. In the current work, we examined the effects of activation and inactivation of MCH system on cilia lengths by using different experimental models and methodologies, including organotypic brain slice cultures from rat prefrontal cortex (PFC) and caudate-putamen (CPu), in vivo pharmacological (MCHR1 agonist and antagonist GW803430), germline and conditional genetic deletion of MCHR1 and MCH, optogenetic, and chemogenetic (designer receptors exclusively activated by designer drugs (DREADD)) approaches. We found that stimulation of MCH system either directly through MCHR1 activation or indirectly through optogenetic and chemogenetic-mediated excitation of MCH-neuron, caused cilia shortening, detected by the quantification of the presence of ADCY3 protein, a known primary cilia marker. In contrast, inactivation of MCH signaling through pharmacological MCHR1 blockade or through genetic manipulations - germline deletion of MCHR1 and conditional ablation of MCH neurons - induced cilia lengthening. Our study is the first to uncover the causal effects of the MCH system in the regulation of the length of brain neuronal primary cilia. These findings place MCH system at a unique position in the ciliary signaling in physiological and pathological conditions and implicate MCHR1 present at primary cilia as a potential therapeutic target for the treatment of pathological conditions characterized by impaired primary cilia function associated with the modification of its length. Show less

Epicardial adipose tissue (EAT) is known to affect atherosclerosis and coronary artery disease (CAD) pathogenesis, persistently releasing pro-inflammatory adipokines that affect the myocardium and coronary arteries. Angiopoietin-like 4 (ANGPTL4) is a protein secreted from adipose tissue and plays a critical role in the progression of atherosclerosis. Here, the expression of ANGPTL4 in EAT was investigated in CAD subjects. Thirty-four consecutive patients (13 patients with significant CAD; 21 patients without CAD) undergoing elective open-heart surgery were recruited. EAT and pericardial fluid were obtained at the time of surgery. mRNA expression and ANGPTL4 and IL-1β levels were evaluated by qRT-PCR and ELISA. The expression of ANGPTL4 (p = 0.0180) and IL-1β (p < 0.0001) in EAT significantly increased in the CAD group compared to that in the non-CAD group and positively correlated (p = 0.004). Multiple regression analysis indicated that CAD is a contributing factor for ANGPTL4 expression in EAT. IL-1β level in the pericardial fluid was significantly increased in patients with CAD (p = 0.020). Moreover, the expression of ANGPTL4 (p = 0.004) and IL-1β (p < 0.001) in EAT was significantly increased in non-obese patients with CAD. In summary, ANGPTL4 expression in EAT was increased in CAD patients. Show less

All clinical BACE1-inhibitor trials for the treatment of Alzheimer's Disease (AD) have failed due to insufficient efficacy or side effects like worsening of cognitive symptoms. However, the scientific evidence to date suggests that BACE1-inhibition could be an effective preventative measure if applied prior to the accumulation of amyloid-beta (Aβ)-peptide and resultant impairment of synaptic function. Preclinical studies have associated BACE1-inhibition-induced cognitive deficits with decreased dendritic spine density. Therefore, we investigated dose-dependent effects of BACE1-inhibition on hippocampal dendritic spine dynamics in an APP knock-in mouse line for the first time. We conducted Show less

We aimed to determine the similarities and differences in the roles of genic and regulatory copy number variations (CNVs) in bipolar disorder (BD), schizophrenia (SCZ), and autism spectrum disorder (ASD). Based on high-resolution CNV data from 8708 Japanese samples, we performed to our knowledge the largest cross-disorder analysis of genic and regulatory CNVs in BD, SCZ, and ASD. In genic CNVs, we found an increased burden of smaller (<100 kb) exonic deletions in BD, which contrasted with the highest burden of larger (>500 kb) exonic CNVs in SCZ/ASD. Pathogenic CNVs linked to neurodevelopmental disorders were significantly associated with the risk for each disorder, but BD and SCZ/ASD differed in terms of the effect size (smaller in BD) and subtype distribution of CNVs linked to neurodevelopmental disorders. We identified 3 synaptic genes (DLG2, PCDH15, and ASTN2) as risk factors for BD. Whereas gene set analysis showed that BD-associated pathways were restricted to chromatin biology, SCZ and ASD involved more extensive and similar pathways. Nevertheless, a correlation analysis of gene set results indicated weak but significant pathway similarities between BD and SCZ or ASD (r = 0.25-0.31). In SCZ and ASD, but not BD, CNVs were significantly enriched in enhancers and promoters in brain tissue. BD and SCZ/ASD differ in terms of CNV burden, characteristics of CNVs linked to neurodevelopmental disorders, and regulatory CNVs. On the other hand, they have shared molecular mechanisms, including chromatin biology. The BD risk genes identified here could provide insight into the pathogenesis of BD. Show less

Interleukin-27 is a pleiotropic cytokine whose functions during bacterial infections remain controversial, and its role in patients with S. aureus osteomyelitis is unknown. To address this knowledge gap, we completed a clinical study and observed elevated serum IL-27 levels (20-fold higher, P < 0.05) in patients compared with healthy controls. Remarkably, IL-27 serum levels were 60-fold higher in patients immediately following septic death than in uninfected patients (P < 0.05), suggesting a pathogenic role of IL-27. To test this hypothesis, we evaluated S. aureus osteomyelitis in WT and IL-27Rα Show less

Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous neuromuscular disorder characterized by muscle weakness and caused by mutations in more than 35 different genes. This condition should not be overlooked as a subset of patients with CMS are treatable. However, the diagnosis of CMS is often difficult due to the broad variability in disease severity and course. A five-year-old boy without remarkable family history was born with marked general muscle hypotonia and weakness, respiratory insufficiency, anomalies, and multiple joint contractures. Congenital myopathy was suspected based upon type 1 fiber predominance on muscle biopsy. However, he was diagnosed with CMS at age 4 years when his ptosis and ophthalmoplegia were found to be improved by edrophonium chloride and repetitive nerve stimulation showed attenuation of compound muscle action potentials. An exome sequencing identified a compound heterozygous missense variant of c.737C > T (p.A246V) and a novel intronic insertion c.1166 + 4₁₁₆₆ + 5insAAGCCCACCAC in RAPSN. RT-PCR analysis which showed the skipping of exon 7 in a skeletal muscle sample confirmed that the intronic insertion was pathogenic. His myasthenic symptoms were remarkably improved by pyridostigmine. The patient's diagnosis of CMS was confirmed by exome sequencing, and RT-PCR revealed that the skipping of exon 7 in RAPSN was caused by a novel intronic insertion. The genetic information uncovered in this case should therefore be added to the collection of tools for diagnosing and treating CMS. Show less

We investigated whether BMP4, FGF8, and/or WNT3a on neural crest-like cells (NCLC) derived from mouse induced pluripotent stem (miPS) cells will promote differentiation of odontoblasts-like cells. After the miPS cells matured into embryonic body (EB) cells, they were cultured in a neural induction medium to produce NCLC. As the differentiation of NCLC were confirmed by RT-qPCR, they were then disassociated and cultured with a medium containing, BMP4, FGF8, and/or WNT3a for 7 and 14 days. The effect of these stimuli on NCLC were assessed by RT-qPCR, ALP staining, and immunocytochemistry. The cultured EB cells presented a significant increase of Snai1, Slug, and Sox 10 substantiating the differentiation of NCLC. NCLC stimulated with more than two stimuli significantly increased the odontoblast markers Dmp-1, Dspp, Nestin, Alp, and Runx2 expression compared to control with no stimulus. The expression of Dmp-1 and Dspp upregulated more when FGF8 was combined with WNT3a. ALP staining was positive in groups containing BMP4 and fluorescence was observed in immunocytochemistry of the common significant groups between Dmp-1 and Dspp. After stimulation, the cell morphology demonstrated a spindle-shaped cells with long projections resembling odontoblasts. Simultaneous BMP4, FGF8, and WNT3a stimuli significantly differentiated NCLC into odontoblast-like cells. Show less

Glucose-dependent insulinotropic polypeptide (GIP) has been reported to have an atheroprotective property in animal models. However, the effect of GIP on macrophage foam cell formation, a crucial step of atherosclerosis, remains largely unknown. We investigated the effects of GIP on foam cell formation of, and Show less

The aim of the present study was to consider whether the ultrastructural features of cardiomyocytes in dilated cardiomyopathy can be used to guide genetic testing. Endomyocardial biopsy and whole-exome sequencing were performed in 32 consecutive sporadic dilated cardiomyopathy patients [51.0 (40.0-64.0) years, 75% men] in initial phases of decompensated heart failure. The predicted pathogenicity of ultrarare (minor allele frequency ≤0.0005), non-synonymous variants was determined using the American College of Medical Genetics guidelines. Focusing on 75 cardiomyopathy-susceptibility and 41 arrhythmia-susceptibility genes, we identified 404 gene variants, of which 15 were considered pathogenic or likely pathogenic in 14 patients (44% of 32). There were five sarcomeric gene variants (29% of 17 variants) found in five patients (16% of 32), involving a variant of MYBPC3 and four variants of TTN. A patient with an MYBPC3 variant showed disorganized sarcomeres, three patients with TTN variants located in the region encoding the A-band domain showed sparse sarcomeres, and a patient with a TTN variant in encoding the I-band domain showed disrupted sarcomeres. The distribution of diffuse myofilament lysis depended on the causal genes; three patients with the same TMEM43 variant had diffuse myofilament lysis near nuclei (P = 0.011), while two patients with different DSP variants had lysis in the peripheral areas of cardiomyocytes (P = 0.033). Derangement patterns of myofilament and subcellular distribution of myofilament lysis might implicate causal genes. Large-scale studies are required to confirm whether these ultrastructural findings are related to the causative genes. Show less

The mTOR pathway is one of the key signal cascades in the pathogenesis of idiopathic pulmonary fibrosis. Previous studies have mainly focused on this pathway in the fibroblasts and/or myofibroblasts, but not in the epithelial cells. In this study, we sought to investigate the role of the mTOR pathway in lung epithelial cells in lung fibrosis. Using Show less

We previously found downregulation of low-density lipoprotein receptor class A domain-containing protein 4 (LDLRAD4), a negative regulator of transforming growth factor (TGF)-β signaling, in glutathione S-transferase placental form (GST-P) expressing ( Show less

Nutrient excess, a major driver of obesity, diminishes hypothalamic responses to exogenously administered leptin, a critical hormone of energy balance. Here, we aimed to identify a physiological signal that arises from excess caloric intake and negatively controls hypothalamic leptin action. We found that deficiency of the gastric inhibitory polypeptide receptor (Gipr) for the gut-derived incretin hormone GIP protected against diet-induced neural leptin resistance. Furthermore, a centrally administered antibody that neutralizes GIPR had remarkable antiobesity effects in diet-induced obese mice, including reduced body weight and adiposity, and a decreased hypothalamic level of SOCS3, an inhibitor of leptin actions. In contrast, centrally administered GIP diminished hypothalamic sensitivity to leptin and increased hypothalamic levels of Socs3. Finally, we show that GIP increased the active form of the small GTPase Rap1 in the brain and that its activation was required for the central actions of GIP. Altogether, our results identify GIPR/Rap1 signaling in the brain as a molecular pathway linking overnutrition to the control of neural leptin actions. Show less

Rett syndrome (RTT) is a characteristic neurological disease presenting with regressive loss of neurodevelopmental milestones. Typical RTT is generally caused by abnormality of methyl-CpG binding protein 2 ( We performed WES on 77 Pathogenic or likely pathogenic single-nucleotide variants in 28 known genes were found in 39 of 77 (50.6%) patients. WES-based CNV analysis revealed pathogenic deletions involving six known genes (including Our study provides a new landscape including additional genetic variants contributing to RTT-like phenotypes, highlighting the importance of comprehensive genetic analysis. Show less

Glucose-dependent insulinotropic polypeptide (GIP) exhibits direct cardiovascular actions in addition to its well-known insulinotropic effect. However, the role of GIP in peripheral artery disease remains unclear. In this study, we evaluated the effects of GIP against peripheral arterial remodeling in mouse models. The genetic deletion of GIP receptor (GIPR) led to exaggerated neointimal hyperplasia after transluminal femoral artery wire injury. Conversely, chronic GIP infusion suppressed neointimal hyperplasia and facilitated endothelial regeneration. The beneficial effects of GIP were abrogated by inhibiting nitric oxide (NO) synthase, suggesting a possible mechanism mediated by NO. In cultured human umbilical vein endothelial cells (HUVECs), GIP elevated cytosolic calcium levels without affecting intracellular cAMP levels. Furthermore, GIP dose-dependently increased NO production, whereas this effect was abolished by inhibiting AMP-activated protein kinase (AMPK). GIP induced AMPK phosphorylation, which was abrogated by inhibiting phospholipase C and calcium-calmodulin-dependent protein kinase kinase but not by adenylate cyclase or liver kinase B1, suggesting the existence of a calcium-mediated GIPR signaling pathway. These effects of GIP were retained in severe hyperglycemic Leprdb/ Leprdb mice and in high-glucose-cultured HUVECs. Overall, we demonstrated the protective effects of GIP against peripheral arterial remodeling as well as the involvement of a calcium-mediated GIPR signaling pathway in vascular endothelial cells. Our findings imply the potential vascular benefits of multiple agonists targeting G protein-coupled receptors, including GIPR, which are under development for the treatment of type 2 diabetes. Show less

Cell polarity is a fundamental property used to generate asymmetry and structure in all cells. Cancer is associated with loss of cell and tissue structure. While observations made in model system such as Drosophila, identify polarity regulators as tumor suppressors that cause inappropriate cell division, studies in mammalian epithelia do not always support such a causative contribution. Our analysis of published cancer dataset shows that many polarity genes, including PARD6B, SCRIB, PRKCI, DLG1, DLG2, DLG5 and LLGL2, are frequently amplified in multiple cancers raising the possibility that mammalian epithelia may have evolved to use polarity proteins in multiple ways where they may have tumor promoting functions. In this review, we reinterpret the published results and propose a modified perspective for the role of polarity regulators in cancer biology. In addition to the traditional form of cell polarity, which is involved establishment of maintenance of normal cell structure and asymmetry, we propose that some mammalian polarity proteins also regulate subcellular polarity (intracellular asymmetry), which can improve cellular fitness to carry out functions such as proliferation, apoptosis, stress adaptation, stemness and organelle biology. Here, we define subcellular polarity and discuss evidence that supports a role for subcellular polarity in biology. Show less

Genome-wide association studies (GWASs) have identified several susceptibility loci for bipolar disorder (BD) and shown that the genetic architecture of BD can be explained by polygenicity, with numerous variants contributing to BD. In the present GWAS (Phase I/II), which included 2964 BD and 61 887 control subjects from the Japanese population, we detected a novel susceptibility locus at 11q12.2 (rs28456, P=6.4 × 10 Show less

To date, mutations in 15 actin- or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts. Show less

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are genetically and phenotypically heterogeneous. Cardiac function is improved after treatment in some cardiomyopathy patients, but little is known about genetic predictors of long-term outcomes and myocardial recovery following medical treatment. To elucidate the genetic basis of cardiomyopathy in Japan and the genotypes involved in prognosis and left ventricular reverse remodeling (LVRR), we performed targeted sequencing on 120 DCM (70 sporadic and 50 familial) and 52 HCM (15 sporadic and 37 familial) patients and integrated their genotypes with clinical phenotypes. Among the 120 DCM patients, 20 (16.7%) had TTN truncating variants and 13 (10.8%) had LMNA variants. TTN truncating variants were the major cause of sporadic DCM (21.4% of sporadic cases) as with Caucasians, whereas LMNA variants, which include a novel recurrent LMNA E115M variant, were the most frequent in familial DCM (24.0% of familial cases) unlike Caucasians. Of the 52 HCM patients, MYH7 and MYBPC3 variants were the most common (12 (23.1%) had MYH7 variants and 11 (21.2%) had MYBPC3 variants) as with Caucasians. DCM patients harboring TTN truncating variants had better prognosis than those with LMNA variants. Most patients with TTN truncating variants achieved LVRR, unlike most patients with LMNA variants. Show less

Apolipoprotein A-IV (apoA-IV) in the brain potently suppresses food intake. However, the mechanisms underlying its anorexigenic effects remain to be identified. We first examined the effects of apoA-IV on cellular activities in hypothalamic neurons that co-express agouti-related peptide (AgRP) and neuropeptide Y (NPY) and in neurons that express pro-opiomelanocortin (POMC). We then compared anorexigenic effects of apoA-IV in wild-type mice and in mutant mice lacking melanocortin 4 receptors (MC4Rs; the receptors of AgRP and the POMC gene product). Finally, we examined expression of apoA-IV in mouse hypothalamus and quantified its protein levels at fed versus fasted states. We demonstrate that apoA-IV inhibited the firing rate of AgRP/NPY neurons. The decreased firing was associated with hyperpolarized membrane potential and decreased miniature excitatory postsynaptic current. We further used c-fos immunoreactivity to show that intracerebroventricular (i.c.v.) injections of apoA-IV abolished the fasting-induced activation of AgRP/NPY neurons in mice. Further, we found that apoA-IV depolarized POMC neurons and increased their firing rate. In addition, genetic deletion of MC4Rs blocked anorexigenic effects of i.c.v. apoA-IV. Finally, we detected endogenous apoA-IV in multiple neural populations in the mouse hypothalamus, including AgRP/NPY neurons, and food deprivation suppressed hypothalamic apoA-IV protein levels. Our findings support a model where central apoA-IV inhibits AgRP/NPY neurons and activates POMC neurons to activate MC4Rs, which in turn suppresses food intake. Show less

Basaloid squamous cell carcinoma (BSCC) is a rare and poorly differentiated variant of typical squamous cell carcinoma, and is characterised in part by activation of the Wnt signalling pathway. We previously demonstrated that constitutive activation of the Wnt signalling pathway by epigenetic silencing of secreted frizzled-related protein 4 (SFRP4) is observed in this tumour. Increasing evidence shows that the Wnt signalling pathway cross-talks with other developmental pathways, including the Hedgehog (HH) pathway. The HH pathway is stimulated by inactivating mutations of PTCH1, which have a well-described oncogenic role in basal cell carcinoma (BCC) of the skin. We employed polymerase chain reaction followed by direct sequencing to detect inactivating mutations of PTCH1 using archival tissue samples of 30 oesophageal BSCCs. The frequency of PTCH1 mutation was compared to that of Wnt component genes that we reported previously. We found PTCH1 mutations in 53.3% (16/30) of cases, revealing T1195S as a hotspot mutation. This frequency is quite high for cancers other than BCC of the skin, and PTCH1 mutations were almost mutually exclusive with mutations in APC, Axin1 and Axin2. Considering the fact that activation of Wnt signalling via down-regulation of APC and SFRP5 due to promoter methylation is observed in BCC of the skin, Wnt signalling activation in oesophageal BSCC might be a secondary effect of the PTCH1-inactivating mutations. These findings suggest that the HH and Wnt pathways coordinately contribute to tumourigenesis in oesophageal BSCC. Furthermore, this study provides a potential therapeutic application for HH pathway inhibitors in oesophageal BSCC with highly malignant potential. Show less

The use of lavender oil (LO)--a commonly, used oil in aromatherapy, with well-defined volatile components linalool and linalyl acetate--in non-traditional medicine is increasing globally. To understand and demonstrate the potential positive effects of LO on the body, we have established an animal model in this current study, investigating the orally administered LO effects genome wide in the rat small intestine, spleen, and liver. The rats were administered LO at 5 mg/kg (usual therapeutic dose in humans) followed by the screening of differentially expressed genes in the tissues, using a 4×44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA) in conjunction with a dye-swap approach, a novelty of this study. Fourteen days after LO treatment and compared with a control group (sham), a total of 156 and 154 up (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes, 174 and 66 up- (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes, and 222 and 322 up- (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes showed differential expression at the mRNA level in the small intestine, spleen and liver, respectively. The reverse transcription-polymerase chain reaction (RT-PCR) validation of highly up- and down-regulated genes confirmed the regulation of the Papd4, Lrp1b, Alb, Cyr61, Cyp2c, and Cxcl1 genes by LO as examples in these tissues. Using bioinformatics, including Ingenuity Pathway Analysis (IPA), differentially expressed genes were functionally categorized by their Gene Ontology (GO) and biological function and network analysis, revealing their diverse functions and potential roles in LO-mediated effects in rat. Further IPA analysis in particular unraveled the presence of novel genes, such as Papd4, Or8k5, Gprc5b, Taar5, Trpc6, Pld2 and Onecut3 (up-regulated top molecules) and Tnf, Slc45a4, Slc25a23 and Samt4 (down-regulated top molecules), to be influenced by LO treatment in the small intestine, spleen and liver, respectively. These results are the first such inventory of genes that are affected by lavender essential oil (LO) in an animal model, forming the basis for further in-depth bioinformatics and functional analyses and investigation. Show less

Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions. iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed. Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions. iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation. Show less

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly. Show less

Basaloid squamous cell carcinoma (BSCC) of the esophagus is a rare variant of typical squamous cell carcinoma (SCC) associated with poor survival. A characteristic feature is nuclear accumulation of β-catenin, without a mutation of the gene. We studied the methylation status of Wnt antagonist genes, such as secreted frizzled-related protein (sFRP) gene family members, Wnt inhibitory factor-1 (WIF-1), Dickkopf-1 (Dkk-1), and human Dapper protein-1 (HDPR-1), and alterations of the APC, Axin1, and Axin2 genes in 30 cases of esophageal BSCC. β-catenin and sFRP (sFRP-1, sFRP-2, sFRP-4, sFRP-5) protein expression was examined by immunohistochemistry. APC, Axin1, and Axin2 gene mutations were detected in 3, 2, and 2 cases, respectively, and 6 cases (20 %) harbored at least 1 alteration in these genes. Methylation of the sFRP-2 promoter region was observed in all cases, and methylation was frequent in sFRP-1 and sFRP-5, but infrequent in Dkk-1, WIF-1, sFRP-4, and HDPR-1. sFRP-2 expression was almost completely absent in 25 cases (83 %), consistent with the methylation status. Nuclear accumulation of β-catenin was observed in all cases. sFRP-5 expression was associated with a low nuclear β-catenin labeling index. These results show that sFRP-2 is a target gene of hypermethylation in esophageal BSCC and suggest that sFRP-2 might contribute to BSCC tumorigenesis through the Wnt/β-catenin signaling pathway. Show less

Gastric neoplasia of chief cell-predominant type (GN-CCP) has been reported as a new, rare variant of gastric tumor. GN-CCPs were defined as tumors consisting of irregular anastomosing glands of columnar cells mimicking chief cells of fundic gland with nuclear atypia and prolapse-type submucosal involvement. We comparatively evaluated clinicopathologic features between 31 GN-CCPs and 130 cases of conventional gastric adenocarcinoma invading into submucosa (CGA-SM) in addition to nuclear β-catenin immunolabeling and direct sequencing of members of the Wnt/β-catenin pathway, CTNNB1, APC, and AXIN, in a subset of these tumors. GN-CCP presented as small protruded lesions located in the upper third of the stomach, with minimal involvement into the submucosa and rare lymphovascular invasion. None of the lesions have demonstrated a recurrence of disease or metastasis on follow-up. Nuclear β-catenin immunolabeling was higher in GN-CCP (labeling index [LI]: median, 19.3%; high expresser [LI >30%], 7/27 cases [26%]) than CGA-SM (median LI, 14.7%; high expresser, 1/19 cases [6%]). Missense mutation of APC was observed in 1 GN-CCP but not CGA-SM. Missense or nonsense mutations of CTNNB1 and AXIN1 were higher in GN-CCPs (14.8%, both) than CGA-SMs (5.3%, both). Missense mutations of AXIN2 were higher in GN-CCPs (25.9%) than in CGA-SMs (10.5%). Overall, 14 (51.9%) of 27 GN-CCPs and 5 (26.3%) of 19 CGA-SM cases harbored at least 1 of these gene mutations. In conclusion, GN-CCPs as a unique variant of nonaggressive tumor are characterized by nuclear β-catenin accumulation and mutation of CTNNB1 or AXIN gene, suggesting activation of the Wnt/β-catenin pathway. Show less