A missense variant in PER2 is associated with delayed sleep-wake phase disorder in a Japanese population.

Delayed sleep-wake phase disorder (DSWPD) is a subtype of circadian rhythm sleep-wake disorders, and is characterized by an inability to fall asleep until late at night and wake up at a socially acceptable time in the morning. The study aim was to identify low-frequency nonsense and missense variants that are associated with DSWPD. Candidate variants in circadian rhythm-related genes were extracted by integration of genetic variation databases and in silico assessment. We narrowed down the candidates to six variants. To examine whether the six variants are associated with DSWPD, we performed an association study in 236 Japanese patients with DSWPD and 1436 controls. A low-frequency missense variant (p.Val1205Met) in PER2 showed a significant association with DSWPD (2.5% in cases and 1.1% in controls, P = 0.026, odds ratio (OR) = 2.32). The variant was also associated with idiopathic hypersomnia known to have a tendency toward phase delay (P = 0.038, OR = 2.07). PER2 forms a heterodimer with CRY, and the heterodimer plays an important role in the regulation of circadian rhythms. Val1205 is located in the CRY-binding domain of PER2 and was hypothesized to interact with CRY. The p.Val1205Met substitution could be a potential genetic marker for DSWPD.

Lack of association between PER3 variable number tandem repeat and circadian rhythm sleep-wake disorders.

Circadian rhythm sleep-wake disorders (CRSWDs) are characterized by disturbed sleep-wake patterns. We genotyped a PER3 variable number tandem repeat (VNTR) in 248 CRSWD individuals and 925 controls and found no significant association between the VNTR and CRSWDs or morningness-eveningness (diurnal) preferences in the Japanese population. Although the VNTR has been associated with circadian and sleep phenotypes in some other populations, the polymorphism may not be a universal genetic marker.

Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+ signaling in human retinal pigment epithelial cells.

Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+ signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks.

[The Outpatient Clinic and Rehabilitation Program Specialized in Adult Developmental Disorders].

The rehabilitation program has been conducted at our psychiatric clinic for depressive patients who are absent from work, with the aim of assisting them to return to work. We have noticed that a substantial number of the patients have traits of developmental disorders, which contribute to chronicity and/or recurrence of depression. Therefore, we have recently created a new rehabilitation program in addition to the specialty outpatient clinic and peer support group. All these programs specialize in treating adult patients with mild developmental disorders [mostly autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD)]. Since then, we have investigated a lot of depressive patients whose ASD symptoms have been identified for the first time in their life. Symptoms were first noted after they started work where they experienced impaired social functioning because the social demands were higher than those at schools. To assist patients with their goals of improving symptoms and stabilizing social functions, it is valid to evaluate whether the autistic traits cause mental stress and impairment during occupational functioning, even if the diagnosis of ASD is not definitive or the symptoms are below the diagnostic threshold for ASD. The profile of ASD symptoms is different for each patient, and therefore personalized support is essential.

Screening of clock gene polymorphisms demonstrates association of a PER3 polymorphism with morningness-eveningness preference and circadian rhythm sleep disorder.

A system of self-sustained biological clocks controls the 24-h rhythms of behavioral and physiological processes such as the sleep-wake cycle. The circadian clock system is regulated by transcriptional and translational negative feedback loops of multiple clock genes. Polymorphisms in circadian clock genes have been associated with morningness-eveningness (diurnal) preference, familial advanced sleep phase type (ASPT), and delayed sleep phase type (DSPT). We genotyped single-nucleotide polymorphisms in circadian clock genes in 182 DSPT individuals, 67 free-running type (FRT) individuals, and 925 controls. The clock gene polymorphisms were tested for associations with diurnal preference and circadian rhythm sleep disorder (CRSD) phenotypes. The PER3 polymorphism (rs228697) was significantly associated with diurnal preference and the FRT phenotype. The minor allele of rs228697 was more prevalent in evening types than in morning types (sex-adjusted odds ratio (OR), 2.483, Bonferroni-corrected P = 0.012) and in FRT individuals compared with the controls (age- and sex-adjusted OR, 2.021, permutated P = 0.017). Our findings support the notion that PER3 polymorphisms could be a potential genetic marker for an individual's circadian and sleep phenotypes.

Analysis of the molecular pathophysiology of sleep disorders relevant to a disturbed biological clock.

Genetic studies have revealed several clock gene variations/mutations involved in the manifestation of sleep disorders or interindividual differences in sleep-wake patterns, but only part of the genetic risk can be explained by the gene variations/mutations identified to date. Recent progress in research into circadian rhythm generation has provided efficient tools for eliciting the molecular basis of clock-relevant sleep disorders, complementing traditional genetic analysis. While the human master clock resides in the suprachiasmatic nucleus of the hypothalamus (central clock), peripheral tissue cells also generate self-sustained circadian oscillations of clock gene expression (peripheral clock), enabling estimation of individual human clock properties through a single collection of skin fibroblasts or venous blood cells. Some of the established cell lines exhibit autonomous circadian oscillations of clock gene expression, and introduction of clock gene variations into these cell lines by gene targeting makes it possible to investigate changes in the circadian phenotype induced by these variations/mutations without the need for generating transgenic animals. Estimation of human clock properties using peripheral tissue cells, in addition to genetic analysis, will facilitate comprehensive explication of the genetic risk of a variety of disorders relevant to biological clock disturbances, including sleep disorders, mood disorders, and metabolic diseases.

Plasma atrial natriuretic peptide is an early diagnosis and disease severity marker of myxomatous mitral valve disease in dogs.

The aim of this study was to retrospectively assess the clinical usefulness of plasma atrial natriuretic peptide (ANP) concentrations for determining the severity of myxomatous mitral valve disease (MMVD) in dogs. Plasma ANP levels were found to be significantly higher in dogs with MMVD compared to healthy dogs, and plasma ANP levels increased significantly in dogs with progressive heart failure. In dogs with MMVD, stepwise regression analysis revealed that the left atrium/aorta ratio and fractional shortening could be used to predict the plasma ANP concentration. These results indicated that plasma ANP rose with an increase in the volume overload of the left side of the heart. Plasma ANP discriminated cardiomegaly from non-cardiomegaly caused by asymptomatic MMVD. We conclude, therefore, that plasma ANP concentrations may be a clinically useful tool for early diagnosis of asymptomatic MMVD in dogs.

Mitral valve repair under cardiopulmonary bypass in small-breed dogs: 48 cases (2006-2009).

OBJECTIVE: To determine whether mitral valve repair (MVR) under cardiopulmonary bypass would be an effective treatment for mitral regurgitation in small-breed dogs. DESIGN: Retrospective case series. ANIMALS: 48 small-breed dogs (body weight, 1.88 to 4.65 kg [4.11 to 10.25 lb]; age, 5 to 15 years) with mitral regurgitation that underwent surgery between August 2006 and August 2009. PROCEDURES: Cardiopulmonary bypass was performed with a cardiopulmonary bypass circuit. After induction of cardiac arrest, a mitral annuloplasty was performed, and the chordae tendineae were replaced with expanded polytetrafluoroethylene chordal prostheses. After closure of the left atrium and declamping to restart the heart, the thorax was closed. RESULTS: Preoperatively, cardiac murmur was grade 3 of 6 to 6 of 6, thoracic radiography showed cardiac enlargement (median vertebral heart size, 12.0 vertebrae; range, 9.5 to 14.5 vertebrae), and echocardiography showed severe mitral regurgitation and left atrial enlargement (median left atrium-to-aortic root ratio, 2.6; range, 1.7 to 4.0). 45 of 48 dogs survived to discharge. Three months after surgery, cardiac murmur grade was reduced to 0/6 to 3/6, and the heart shadow was reduced (median vertebral heart size, 11.1 vertebrae, range, 9.2 to 13.0 vertebrae) on thoracic radiographs. Echocardiography confirmed a marked reduction in mitral regurgitation and left atrium-to-aortic root ratio (median, 1.7; range, 1.0 to 3.0). CONCLUSIONS AND CLINICAL RELEVANCE: We successfully performed MVR under cardiopulmonary bypass in small-breed dogs, suggesting this may be an effective surgical treatment for dogs with mitral regurgitation. Mitral valve repair with cardiopulmonary bypass can be beneficial for the treatment of mitral regurgitation in small-breed dogs.

An association analysis of Per2 with panic disorder in the Japanese population.

Panic disorder (PD) is a severe and chronic psychiatric disorder, with genetic components underlying in its etiology. The PERIOD2 (Per2) gene has been reported to be associated with familial advanced sleep phase syndrome. Considering the high frequency of sleep disturbance in PD, Per2 may be a candidate gene for PD. Therefore, we conducted a two-stage case-control association study in the Japanese population. In the first screening sample of 203 patients and 409 controls, we investigated three single-nucleotide polymorphisms in Per2. We found a potential association in the screening sample (rs2304672, genotype P=0.046, uncorrected), whereas we could not replicate the association in the second sample of 460 patients and 460 controls. Our results suggest that Per2 may not have a major role in the pathogenesis of PD in the Japanese population.

Surgical closure of an atrial septal defect using cardiopulmonary bypass in a cat.

OBJECTIVE: To describe surgical repair of a large atrial septal defect (ASD) in a cat. STUDY DESIGN: Clinical report. ANIMAL: A 3-year-old, 3.3 kg, intact male Japanese domestic short-haired cat. METHODS: A 10.2-mm-diameter ASD detected by echocardiography was surgically corrected because pulmonary vascular resistance-to-systemic vascular resistance ratio (Qp /Qs ) was 3.2. Using cardiopulmonary bypass (CPB), open surgical repair was achieved with an expanded polytetrafluoroethylene (e-PTFE) graft. The priming volume of the CPB circuit was minimized by cutting the CPB tubing, and partially replacing the priming fluid with whole cat blood. To prevent hemodilution associated with use of cardioprotective agents, surgery was performed on the beating heart. RESULTS: At 1-year echocardiographic evaluation, the repair was intact, and at 3 years, the cat was alive without need of medication. CONCLUSIONS: Large ASD in a cat can be repaired using e-PTFE under CPB.

Surgical repair of a complete endocardial cushion defect in a dog.

OBJECTIVE: To describe surgical repair of a complete endocardial cushion defect (ECD) in a dog. STUDY DESIGN: Clinical report. ANIMAL: A 5-month-old, 9.2 kg male Shetland sheepdog. METHODS: Echocardiographic examination revealed an ostium primum atrial septal defect (ASD), an inlet ventricular septal defect (VSD), mitral regurgitation (MR) and tricuspid regurgitation (TR), and a complete ECD was diagnosed. Surgical correction was performed using cardiopulmonary bypass (CPB) via right atriotomy. A polytetrafluoroethylene (PTFE) patch was secured along the margin of the inlet VSD using simple continuous suture, then the cleft in the septal mitral leaflet was sutured. Similarly, the cleft in the septal tricuspid leaflets was sutured. To complete inlet VSD closure, the VSD patch was secured to these sutured leaflets by simple continuous suture. Another PTFE patch was used to close the ostium primum ASD. RESULT: After surgery, MR, TR, and interventricular shunting were decreased. The dog was alive 6 years and 5 months after the surgery with no evidence of an interventricular shunt, TR, or other clinical signs. CONCLUSIONS: Complete ECD in a dog was corrected using a 2-patch technique under CPB.

Self-sustained circadian rhythm in cultured human mononuclear cells isolated from peripheral blood.

Disturbed circadian rhythmicity is associated with human diseases such as sleep and mood disorders. However, study of human endogenous circadian rhythm is laborious and time-consuming, which hampers the elucidation of diseases. It has been reported that peripheral tissues exhibit circadian rhythmicity as the suprachiasmatic nucleus-the center of the biological clock. We tried to study human circadian rhythm using cultured peripheral blood mononuclear cells (PBMCs) obtained from a single collection of venous blood. Activated human PBMCs showed self-sustained circadian rhythm of clock gene expression, which indicates that they are useful for investigating human endogenous circadian rhythm.

Introduction of tau mutation into cultured Rat1-R12 cells by gene targeting, using recombinant adeno-associated virus vector.

We aim to develop a cultured cell model, to serve as a system with which the altered circadian phenotypes produced by the clock gene variations could be studied in vitro. Tau mutation, which shortens the circadian period of hamsters and mice, was introduced into the CK1epsilon locus of cultured Rat1-R12 cells by gene targeting mediated by a recombinant adeno-associated virus (rAAV) vector. After transduction of Rat1-R12 cells with rAAV, about 0.14% of the drug-resistant cells underwent gene targeting at CK1epsilon locus. Of the three clones isolated, only one carried the targeted allele of tau mutation and two carried the targeted wild-type allele. The clone with the targeted tau mutant allele exhibited a significantly shorter circadian period compared to the clone with targeted wild-type allele. rAAV-mediated gene targeting in cultured somatic cells is a convenient and powerful tool for analyzing the phenotypic outcome of clock gene variations, and for elucidating the pathogenesis of the disorders associated with abnormal circadian rhythmicity.

Continuous observation of rabbit preimplantation embryos in vitro by using a culture device connected to a microscope.

We developed a compact culture device that maintains developing embryos in vitro under constant temperature and CO(2) concentration. Using this device, we cultured rabbit embryos from the pronuclear stage to the hatched blastocyst stage and recorded their development digitally for 7 d. Recorded images were converted to a movie, and the developmental movement of individual embryos was analyzed. With this culture system, we can observe embryonic development in a suitable environment continuously for several days; similar long-term observation is not possible in the conventional system. The proportion of embryos that developed from the pronuclear stage to the blastocyst stage was the same in the new system (73.1%; 38 of 52) as in the conventional (control) system (77.6%; 38 of 49). Compaction of embryos occurred from the 8-cell to the morula stage at 32.5 +/- 0.71 h after insemination. The time of blastocyst formation (77.2 +/- 3.2 h after insemination) varied somewhat between embryos. Average hatching time was 98.7 +/- 4.4 h after mating. Therefore, the cleavage, blastomere movement, and hatching processes of blastocysts can be followed clearly and recorded by using this new culture system.

The influence of gender on cardiac fibrosis induced by sympathetic stimulation.

We examined the influence of sex steroids on cardiac effects of sympathetic agents in mice. The mice were divided into four groups: males, neutered males (N-males), females, and neutered females (N-females). Dobutamine (DOB; 2.5, 5.0, 10 microg/kg/min) or isoproterenol (ISO; 0.01, 0.02, 0.04 microg/kg/min) were given intravenously to compare the fractional shortening (FS). These mice received isoproterenol twice daily at a dose of 7.5 microg/g/day for 3 weeks. The rate of cardiac fibrosis was evaluated pathologically with Azan stain after 3 weeks of ISO. DOB and ISO significantly increased the FS in the male group, compared with other groups. There was no significant difference in FS between the female and N-female groups. Cardiac fibrosis significantly increased in the male group, compared with the N-male group. The female and N-female groups had increased cardiac fibrosis, compared with the male and N-male groups. These findings suggest that testosterone is one of the factors of modulation of the response to the sympathetic nervous system. Further study is needed to clarify the relationships between female sex steroids and cardiac fibrosis.

Establishment of human cell lines showing circadian rhythms of bioluminescence.

We have established human retinal pigment epithelial cell lines stably expressing the luciferase gene, driven by the human Bmal1 promoter, to obtain human-derived cells that show circadian rhythms of bioluminescence after dexamethasone treatment. The average circadian period of bioluminescence for the obtained clones was 24.07+/-0.48 h. Lithium (10 mM) in the medium significantly lengthened the circadian period of bioluminescence, which is consistent with previous reports, while 2 mM or 5 mM lithium had no effect. This is the first report on the establishment of human-derived cell lines that proliferate infinitely and show circadian rhythms of bioluminescence, and also the first to investigate the effects of low-dose lithium on the circadian rhythms of human-derived cells in vitro. The established cells will be useful for various in vitro studies of human circadian rhythms and for the development of new therapies for human disorders related to circadian rhythm disturbances.

Evaluation of the renin-angiotensin system in cardiac tissues of cats with pressure-overload cardiac hypertrophy.

OBJECTIVE: To clarify regulation of the renin-angiotensin (RA) system in cardiac tissues by measuring angiotensin-converting enzyme (ACE) and chymase activities in cats with pressure-overload cardiac hypertrophy. ANIMALS: 13 adult cats. PROCEDURES: Pressure-overload cardiac hypertrophy was induced by coarctation of the base of the ascending aorta in 6 cats, and 7 cats served as untreated control animals. Cats were examined before and 3 months and 2 years after surgery. Two years after surgery, cardiac hypertrophy was confirmed by echocardiography, and the blood pressure gradient was measured at the site of constriction. Cats were euthanized, and ACE and chymase activities were measured in cardiac tissues. RESULTS: Mean +/- SD pressure gradient across the aortic constriction was 63 +/- 6 mm Hg. Chymase activity predominated (75% to 85%) in the RA system of the cardiac tissues of cats. Fibrosis in the wall of the left ventricle was detected in cats with hypertrophy, and fibrosis of the papillary muscle was particularly evident. CONCLUSIONS AND CLINICAL RELEVANCE: Chronic pressure overload on the heart of cats can activate the RA system in cardiac tissues. A local increase in angiotensin II was one of the factors that sustained myocardial remodeling.

[Functional polymorphisms in clock genes and circadian rhythm sleep disorders].

Polymorphisms in clock genes induce circadian rhythm sleep disorders. Mutations in Per2 gene (S662G) or Casein Kinasel delta (CK16) gene (T44A) cause Familial advanced sleep phase syndrome. Missense polymorphisms in Per3 (V647G) and CK1e (S408N) genes increase or decrease the risk of developing delayed sleep phase syndrome. All of these polymorphisms seem to affect the phosphorylation of the clock proteins. Some of the polymorphisms in CK1, which shows reduced enzyme activity in vitro, induced increased phosphorylation of PER proteins in in vivo assays. Careful attention should be paid to analyze the complex system composed of feedback loops, such as the biological clock.