Regulation of headache response and transcriptomic network by the trigeminal ganglion clock.

OBJECTIVE: To characterize the circadian features of the trigeminal ganglion in a mouse model of headache. BACKGROUND: Several headache disorders, such as migraine and cluster headache, are known to exhibit distinct circadian rhythms of attacks. The circadian basis for these rhythmic pain responses, however, remains poorly understood. METHODS: We examined trigeminal ganglion ex vivo and single-cell cultures from Per2::LucSV reporter mice and performed immunohistochemistry. Circadian behavior and transcriptomics were investigated using a novel combination of trigeminovascular and circadian models: a nitroglycerin mouse headache model with mechanical thresholds measured every 6 h, and trigeminal ganglion RNA sequencing measured every 4 h for 24 h. Finally, we performed pharmacogenomic analysis of gene targets for migraine, cluster headache, and trigeminal neuralgia treatments as well as trigeminal ganglion neuropeptides; this information was cross-referenced with our cycling genes from RNA sequencing data to identify potential targets for chronotherapy. RESULTS: The trigeminal ganglion demonstrates strong circadian rhythms in both ex vivo and single-cell cultures, with core circadian proteins found in both neuronal and non-neuronal cells. Using our novel behavioral model, we showed that nitroglycerin-treated mice display circadian rhythms of pain sensitivity which were abolished in arrhythmic Per1/2 double knockout mice. Furthermore, RNA-sequencing analysis of the trigeminal ganglion revealed 466 genes that displayed circadian oscillations in the control group, including core clock genes and clock-regulated pain neurotransmitters. In the nitroglycerin group, we observed a profound circadian reprogramming of gene expression, as 331 of circadian genes in the control group lost rhythm and another 584 genes gained rhythm. Finally, pharmacogenetics analysis identified 10 genes in our trigeminal ganglion circadian transcriptome that encode target proteins of current medications used to treat migraine, cluster headache, or trigeminal neuralgia. CONCLUSION: Our study unveiled robust circadian rhythms in the trigeminal ganglion at the behavioral, transcriptomic, and pharmacogenetic levels. These results support a fundamental role of the clock in pain pathophysiology. PLAIN LANGUAGE SUMMARY: Several headache diseases, such as migraine and cluster headache, have headaches that occur at the same time each day. We learned that the trigeminal ganglion, an important pain structure in several headache diseases, has a 24-hour cycle that might be related to this daily cycle of headaches. Our genetic analysis suggests that some medications may be more effective in treating migraine and cluster headache when taken at specific times of the day.

The red ruffed lemur, Varecia rubra (É. Geoffroy Saint-Hilaire, 1812): a comparative morphology investigation of lingual papillae and connective tissue cores.

The morphology of the lingual papillae (filiform, foliate, fungiform, and vallate papillae) and the underlying connective tissue core of the red ruffed lemur (Varecia rubra) of a strepsirrhines species were studied using light and scanning electron microscopy. The filiform papillae distributed at the root of the tongue were larger than the structures distributed at the body and apex. Six to eight vallate papillae were arranged in a Y-shape at the border between the lingual body and the lingual root. Foliate papillae were observed at the posterior lateral border of the tongue. Scanning electron microscopy revealed a primary process and numerous auxiliary processes in the epithelial layer of filiform papillae. After epithelial removal, the connective tissue core of the filiform papilla showed several protrusions surrounding an oval-shaped depression that extended slightly posteriorly, and a large, maple-shaped filiform papilla was seen in the posterior portion of the tongue. The connective tissue cores of the fungiform papillae exhibited a longitudinally ridged cylindrical structure. The connective tissue core of the foliate papillae had numerous tubular projections arranged along a groove with a salivary gland conduit at the base. As a Lemuridae species, the appearance of the fungiform and filiform papillae of the red ruffed lemur is similar to that reported in previous studies of the ring-tailed lemur, with some differences, especially in the filiform papillary connective tissue core at the base and tongue body border. These findings suggest the taxonomic and phylogenetic origins of the lemurs as well as the influence of dietary diversity.

The circadian E3 ligase FBXL21 regulates myoblast differentiation and sarcomere architecture via MYOZ1 ubiquitination and NFAT signaling.

Numerous molecular and physiological processes in the skeletal muscle undergo circadian time-dependent oscillations in accordance with daily activity/rest cycles. The circadian regulatory mechanisms underlying these cyclic processes, especially at the post-transcriptional level, are not well defined. Previously, we reported that the circadian E3 ligase FBXL21 mediates rhythmic degradation of the sarcomere protein TCAP in conjunction with GSK-3ß, and Psttm mice harboring an Fbxl21 hypomorph allele show reduced muscle fiber diameter and impaired muscle function. To further elucidate the regulatory function of FBXL21 in skeletal muscle, we investigated another sarcomere protein, Myozenin1 (MYOZ1), that we identified as an FBXL21-binding protein from yeast 2-hybrid screening. We show that FBXL21 binding to MYOZ1 led to ubiquitination-mediated proteasomal degradation. GSK-3ß co-expression and inhibition were found to accelerate and decelerate FBXL21-mediated MYOZ1 degradation, respectively. Previously, MYOZ1 has been shown to inhibit calcineurin/NFAT signaling important for muscle differentiation. In accordance, Fbxl21 KO and MyoZ1 KO in C2C12 cells impaired and enhanced myogenic differentiation respectively compared with control C2C12 cells, concomitant with distinct effects on NFAT nuclear localization and NFAT target gene expression. Importantly, in Psttm mice, both the levels and diurnal rhythm of NFAT2 nuclear localization were significantly diminished relative to wild-type mice, and circadian expression of NFAT target genes associated with muscle differentiation was also markedly dampened. Furthermore, Psttm mice exhibited significant disruption of sarcomere structure with a considerable excess of MYOZ1 accumulation in the Z-line. Taken together, our study illustrates a pivotal role of FBXL21 in sarcomere structure and muscle differentiation by regulating MYOZ1 degradation and NFAT2 signaling.

ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor response to standard therapies and therefore unfavorable clinical outcomes. Better understanding of TNBC and new therapeutic strategies are urgently needed. ROR nuclear receptors are multifunctional transcription factors with important roles in circadian pathways and other processes including immunity and tumorigenesis. Nobiletin (NOB) is a natural compound known to display anticancer effects, and our previous studies showed that NOB activates RORs to enhance circadian rhythms and promote physiological fitness in mice. Here, we identified several TNBC cell lines being sensitive to NOB, by itself or in combination. Cell and xenograft experiments showed that NOB significantly inhibited TNBC cell proliferation and motility in vitro and in vivo. ROR loss- and gain-of-function studies showed concordant effects of the NOB-ROR axis on MDA-MB-231 cell growth. Mechanistically, we found that NOB activates ROR binding to the ROR response elements (RRE) of the IκBα promoter, and NOB strongly inhibited p65 nuclear translocation. Consistent with transcriptomic analysis indicating cancer and NF-κB signaling as major pathways altered by NOB, p65-inducible expression abolished NOB effects, illustrating a requisite role of NF-κB suppression mediating the anti-TNBC effect of NOB. Finally, in vivo mouse xenograft studies showed that NOB enhanced the antitumor efficacy in mammary fat pad implanted TNBC, as a single agent or in combination with the chemotherapy agent Docetaxel. Together, our study highlights an anti-TNBC mechanism of ROR-NOB via suppression of NF-κB signaling, suggesting novel preventive and chemotherapeutic strategies against this devastating disease.

The first-line cluster headache medication verapamil alters the circadian period and elicits sex-specific sleep changes in mice.

Verapamil is the first-line preventive medication for cluster headache, an excruciating disorder with strong circadian features. Whereas second- and third-line preventives include known circadian modulators, such as melatonin, corticosteroids, and lithium, the circadian effects of verapamil are poorly understood. Here, we characterize the circadian features of verapamil using both in vitro and in vivo models. In Per2::LucSV reporter fibroblasts, treatment with verapamil (0.03-10 µM) showed a dose-dependent period shortening of the reporter rhythm which reached a nadir at 1 µM, and altered core clock gene expression at 10 µM. Mouse wheel-running activity with verapamil (1 mg/mL added to the drinking water) also resulted in significant period shortening and activity reduction in both male and female free-running wild-type C57BL6/J mice. The temporal patterns of activity reduction, however, differ between the two sexes. Importantly, piezo sleep recording revealed sexual dimorphism in the effects of verapamil on sleep timing and bout duration, with more pronounced adverse effects in female mice. We also found altered circadian clock gene expression in the cerebellum, hypothalamus, and trigeminal ganglion of verapamil-treated mice. Verapamil did not affect reporter rhythms in ex vivo suprachiasmatic nucleus (SCN) slices from Per2:Luc reporter mice, perhaps due to the exceptionally tight coupling in the SCN. Thus, verapamil affects both peripheral (trigeminal ganglion) and central (hypothalamus and cerebellum) nervous system structures involved in cluster headache pathophysiology, possibly with network effects instead of isolated SCN effects. These studies suggest that verapamil is a circadian modulator in laboratory models at both molecular and behavioral levels, and sex is an important biological variable for cluster headache medications. These observations highlight the circadian system as a potential convergent target for cluster headache medications with different primary mechanisms of action.

Discovery of a Potent, Selective, and Orally Available MTHFD2 Inhibitor (DS18561882) with in Vivo Antitumor Activity.

We report the discovery of a potent and isozyme-selective MTHFD2 inhibitor, DS18561882 (2). Through investigation of the substituents on our tricyclic coumarin scaffold (1,2,3,4-tetrahydrochromeno[3,4-c]pyridin-5-one), MTHFD2 inhibitory activity was shown to be elevated by incorporating an amine moiety at the 8-position and a methyl group at the 7-position of the initial lead 1. X-ray structure analysis revealed that a key interaction for enhanced potency was salt bridge formation between the amine moiety and the diphosphate linker of an NAD+ cofactor. Furthermore, ortho-substituted sulfonamide in place of benzoic acid of 1 significantly improved cell permeability and cell-based growth inhibition against a human breast cancer cell line. The thus-optimized DS18561882 showed the strongest cell-based activity (GI50 = 140 nM) in the class, a good oral pharmacokinetic profile, and thereby tumor growth inhibition in a mouse xenograft model upon oral administration.

Comparative morphology of the lingual papillae and their connective tissue cores in the tongue of the Abyssinian black-and-white colobus (Colobus guereza).

We observed the morphology of the lingual papillae (filiform, fungiform, foliate, and vallate) and their underlying connective tissue cores (CTCs) in Abyssinian black-and-white colobus monkeys using light and scanning electron microscopy. The tongues of both juvenile and senescent individuals were relatively short in the rostro-caudal direction, with a rounded apex. Lingual tori were absent. Numerous filiform papillae were distributed over the entire tongue, except at the lingual root. A pair of foliate papillae was present on both the lateral and caudal margins of the corpus. Three vallate papillae were distributed on the boundary between the caudal part of the body and the root in both juvenile and senescent individuals. Based on scanning electron microscopy observations, the morphologies of the filiform papillae differed between juvenile and senescent individuals. The epithelial surface of juvenile filiform papillae had a main process, but the associated processes were weak and the underlying CTCs displayed immature morphology. In contrast, the epithelial surface of senescent filiform papillae was associated with several accessory processes, and their underlying CTCs consisted of several auxiliary cores that nearly encircled the main core, forming a concavity in the papilla. CTCs of the filiform papillae showed variable morphology. Juvenile filiform CTCs exhibited a rather primitive morphology, resembling those of the hamster, mole, and Cape hyrax while, conversely, despite the basically folivorous diet of the Abyssinian black-and-white colobus, senescent filiform CTCs resembled those found in omnivorous primates, including members of the Callitrichinae and Homoidea, and also those in Carnivora (e.g., Canidae and Felidae).

Gender and Smoking Correlations of Surfactant Lipids and Proteins in the Saliva of Dental Patients.

We sought to determine the effects of smoking on surfactant lipids and proteins in saliva. Levels of sphingomyelin (Sph) phosphatidylcholine (PC) and lyso-PC (LPC) were determined by thin layer chromatography. Levels of surfactant protein A (SP-A) were determined by western analysis using antibodies specific for SP-A. Significance of the results was determined by the student's t-test. The LPC/PC ratio had a tendency to be much higher in smokers compared to nonsmokers. LPC levels were significantly higher in females smokers compared to male smokers. Additionally, levels of SP-A were significantly reduced in females smokers compared to non-smokers. Smoking alters surfactant protein and LPC/PC ratios in saliva. There is a significant difference in the effects in females compared to males. Findings suggest smoking alters the composition of saliva that may reduce protection of the oral cavity, which may explain why women smokers are at greater risk of developing oral mucositis.

Low plasma tryptophan is associated with olfactory function in healthy elderly community dwellers in Japan.

BACKGROUND: Decreased circulating tryptophan (Trp) levels are frequently observed in elderly patients with neurodegenerative disease including Alzheimer's disease. Trp may serve as a potential biomarker for monitoring disease risk in elderly people. We aimed to investigate the association between low plasma Trp levels and olfactory function, which is known to predict age-related diseases including dementia in elderly people. METHODS: A total of 144 healthy elderly Japanese community (≥ 65 years old) dwellers from the Health, Aging and Nutritional Improvement study (HANI study) were the subjects of our analysis. Low Trp levels were classified using the lower limit values of the reference interval according to a previous report. Olfactory function was assessed using a card-type test called Open Essence, which includes 12 odour items that are familiar to Japanese people. The elderly subjects with low circulating Trp levels were compared to a control group with normal plasma Trp levels. RESULTS: We conducted the analyses using 144 people aged 65 years or older (mean age 73.7 ± 5.5 years; 36.1% men). The subjects showed normal serum albumin levels (4.4 ± 0.2 g/dL) and no daily living disabilities. Low plasma Trp levels (low Trp group) were found in 11.1% of the study population. The low Trp group showed a significantly lower correct-answer rate for the items india ink, perfume, curry and sweaty smelling socks than control group (P < 0.05). There was also a significant association between low Trp levels and low olfactory ability, after adjusting for age and sex. CONCLUSIONS: Lower plasma Trp levels were associated with a decrease in olfactory function in functionally competent older individuals. Because olfactory dysfunction predicts age-related diseases, low plasma Trp levels may represent a clinical sign of disease risk in elderly people.

Hematological and pathological features of massive hepatic necrosis in two radiated tortoises (Astrochelys radiata).

Two radiated tortoises (Astrochelys radiata) exhibited anorexia and hypokinesia. In both cases, hematological and serum biochemical examinations revealed high alkaline phosphatase levels, moderately high aspartate aminotransferase levels and white blood cell counts approximately within the normal range. Despite being treated, the tortoises died 9 and 43 days after the first clinical examination. Gross pathological examinations revealed that the livers of both animals were extremely swollen and contained pale yellow necrotic tissue. Histopathological assessment revealed that the livers contained a massive area of hepatic necrosis surrounded by migration of macrophages and multinucleated giant cells. In one of the cases, severe fibrosis was observed. The present study provides reference information for similar cases in the future.

Contributions and source identification of biogenic and anthropogenic hydrocarbons to secondary organic aerosols at Mt. Tai in 2014.

Ambient fine particulate matter (PM2.5) and volatile organic compounds (VOCs) collected at Mt. Tai in summer 2014 were analysed and the data were used to identify the contribution of biogenic and anthropogenic hydrocarbons to secondary organic aerosols (SOA) and their sources and potential source areas in high mountain regions. Compared with those in 2006, the 2014 anthropogenic SOA tracers in PM2.5 aerosols and VOC species related to vehicular emissions exhibited higher concentrations, whereas the levels of biogenic SOA tracers were lower, possibly due to decreased biomass burning. Using the SOA tracer and parameterisation method, we estimated the contributions from biogenic and anthropogenic VOCs, respectively. The results showed that the average concentration of biogenic SOA was 1.08 ± 0.51 µg m-3, among which isoprene SOA tracers were dominant. The anthropogenic VOC-derived SOA were 7.03 ± 1.21 µg m-3 and 1.92 ± 1.34 µg m-3 under low- and high-NOx conditions, respectively, and aromatics made the greatest contribution. However, the sum of biogenic and anthropogenic SOA only contributed 18.1-49.1% of the total SOA. Source apportionment by positive matrix factorisation (PMF) revealed that secondary oxidation and biomass burning were the major sources of biogenic SOA tracers. Anthropogenic aromatics mainly came from solvent use, fuel and plastics combustion and vehicular emissions. However, for > C6 alkanes and cycloalkanes, vehicular emissions and fuel and plastics combustion were the most important contributors. The potential source contribution function (PSCF) identified the Bohai Sea Region (BSR) as the major source area for organic aerosol compounds and VOC species at Mt. Tai.

Extraction of an incisor embedded within the nasal cavity in two guinea pigs.

Oral examination of two guinea pigs revealed that the unilateral incisor was absent. On radiographic examination, the incisor was identified within the nasal cavity in both patients. Under anesthesia in both patients, the skin was incised from the nostril to 1.5 cm proximal, and the premaxilla and part of the maxilla were exposed. The bone was removed using a surgical drill, and the incisor was exposed in the nasal cavity. The root was grasped with forceps and carefully extracted as it was degraded and very fragile. Diagnosis was easy using oral and radiographic examination. In guinea pig patients where an incisor is absent on oral examination, this condition should be considered.

FOLLICULAR CELL CARCINOMA OF THE THYROID GLAND IN THREE CAPTIVE AGED RACCOON DOGS (NYCTEREUTES PROCYONOIDES).

The clinical and histologic features of thyroid carcinoma in raccoon dogs have not been previously reported. Three of four raccoon dogs (Nyctereutes procyonoides) over 8 yr of age at the Nogeyama Zoological Gardens developed thyroid follicular cell carcinomas that were detected at necropsy. The affected raccoon dogs were rescued from the wild and were housed at the Nogeyama Zoological Gardens for 8 yr 8 mo, 8 yr 10 mo, and 10 yr 3 mo, respectively. Although all of them appeared lethargic and developed partial alopecia or desquamation of their skin, they did not display any other specific clinical signs associated with a thyroid lesion. Serum thyroid hormone values were examined in two of the affected raccoon dogs and the average and standard deviation values (free-thyroxin [FT4]: 0.078 ± 0.077 pM/L and 0.062 ± 0.0039 pM/L; free-triiodothyronine [FT3]: 3.261 ± 0.765 pM/L and 3.407 ± 0.919 pM/L) were lower than the reference range (FT4: 0.141 ± 0.117 pM/L; FT3: 5.139 ± 2.412 pM/L) derived from a clinically normal raccoon dog. On necropsy, the thyroid lobes were markedly enlarged bilaterally. Histopathologically, the neoplastic cells in the thyroid gland appeared round or oval and columnar or cuboidal with minimal heteromorphism. Moreover, mostly small (but occasionally large) follicles were identified, and the neoplastic cells had infiltrated into the surrounding capsule and blood vessels. The histopathologic features of the thyroid tumors in the raccoon dogs revealed that the tumors were derived from follicular cells.

A new voluntary blood collection method for the Andean bear (Tremarctos ornatus) and Asiatic black bear (Ursus thibetanus).

Various training methods have been developed for animal husbandry and health care in zoos and one of these trainings is blood collection. One training method, recently widely used for blood collection in Ursidae, requires setting up a sleeve outside the cage and gives access to limited blood collection sites. A new voluntary blood collection method without a sleeve was applied to the Andean bear (Tremarctos ornatus) and Asiatic black bear (Ursus thibetanus) with access to various veins at the same time. The present study evaluated the effectiveness of this new method and suggests improvements. Two Andean and two Asiatic black bears in Yokohama and Nogeyama Zoological Gardens, respectively, were trained to hold a bamboo pipe outside their cages. We could, thereby, simultaneously access superficial dorsal veins, the dorsal venous network of the hand, the cephalic vein from the carpal joint, and an area approximately 10 cm proximal to the carpal joint. This allowed us to evaluate which vein was most suitable for blood collection. We found that the cephalic vein, approximately 10 cm proximal to the carpal joint, was the most suitable for blood collection. This new method requires little or no modification of zoo facilities and provides a useful alternative method for blood collection. It could be adapted for use in other clinical examinations such as ultrasound examination.

High abundances of dicarboxylic acids, oxocarboxylic acids, and α-dicarbonyls in fine aerosols (PM2.5) in Chengdu, China during wintertime haze pollution.

Daytime and nighttime fine aerosol (PM2.5) samples were collected during a haze episode in January 2013 within the urban area of Chengdu, southwest China. Aerosol samples were analyzed for low-molecular-weight homologous dicarboxylic acids, oxocarboxylic acids and α-dicarbonyls, as well as organic carbon and elemental carbon. Concentration ranges of diacids, oxoacids, and α-dicarbonyls were 1,400-5,250, 272-1,380, and 88-220 ng m(-3), respectively. Molecular distributions of diacids (mean 3,388 ± 943 ng m(-3)) were characterized by a predominance of oxalic acid (C2; 1,373 ± 427 ng m(-3)), followed by succinic (C4), terephthalic (tPh), and phthalic (Ph) acids. Such high levels of tPh and Ph were different from those in other Asian cities where malonic acid (C3) is the second or third highest species, mostly owing to significant emissions from coal combustion and uncontrolled waste incineration. High contents of diacids, oxoacids, and α-dicarbonyls were detected on hazy days, suggesting an enhanced emission and/or formation of these organics during such a weather condition. Concentrations of unsaturated aliphatic diacids (e.g., maleic acid) and phthalic acids were higher in nighttime than in daytime. Good positive correlations of C2 with C3, C4, ketomalonic (kC3), pyruvic (Pyr), and glyoxylic (É·C2) acids in daytime suggest secondary production of C2 via the photooxidation of longer chain diacids and É·C2. This study demonstrated that both primary emissions and secondary production are important sources of dicarboxylic acids and related compounds in atmospheric aerosols in the Sichuan Basin.

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa.

Biofilms are communities of surface-attached microbial cells that resist environmental stresses. In this study, we found that low concentrations of ethanol increase biofilm formation in Pseudomonas aeruginosa PAO1 but not in a mutant of it lacking both Psl and Pel exopolysaccharides. Low concentrations of ethanol also increased pellicle formation at the air-liquid interface.

Insufficient colostrum ingestion is a risk factor for polyarthritis and/or phlegmon in hand-reared reticulated giraffes (Giraffa camelopardalis reticulata): 7 cases (2003-2012).

Seven reticulated giraffes were hand-reared at Nogeyama Zoological Gardens, because the dam had agalactia. Six of the 7 calves exhibited polyarthritis and/or phlegmon in the lower legs. However, the cause of the disorder was unclear. The present study reviewed the clinical records of the 7 giraffes, including the type and amount of colostrum ingested during the first 72 hr. The disorder involved the fetlocks and carpal and tarsal joints in 6 of the 7 calves within an average of 8 days of birth. The average amount of fed bovine or powdered colostrum was 0-2.4 l in the first 24 hr and 2.0-6.2 l during the first 72 hr. Insufficient colostrum quantity might be a factor in polyarthritis and/or phlegmon.

cAMP signaling affects irreversible attachment during biofilm formation by Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa responds to environmental changes and regulates its life cycle from planktonic to biofilm modes of growth. The control of cell attachment to surfaces is one of the critical processes that determine this transition. Environmental signals are typically relayed to the cytoplasm by second messenger systems. We here demonstrated that the second messenger, cAMP, regulated the attachment of cells. Our results suggest cAMP inhibited the transition from reversible to irreversible attachment. Further analyses revealed that cell surface hydrophobicity, one of the key factors in cell attachment, was altered by cAMP.

Regional variations and age-related changes detected with magnetic resonance spectroscopy in the brain of healthy dogs.

OBJECTIVE: To investigate age-related and regional differences in estimated metabolite concentrations in the brain of healthy dogs by means of magnetic resonance spectroscopy (MRS). ANIMALS: 15 healthy Beagles. PROCEDURES: Dogs were grouped according to age as young (n = 5; all dogs were 2 months old), adult (5; mean age, 4.5 years), or geriatric (5; all dogs were 12 years old). Imaging was performed by use of a 1.5-T MRI system with T1- and T2-weighted spin-echo and fluid-attenuated inversion recovery sequences. Signal intensity measurements for N-acetyl aspartate, creatine, choline, and lactate-alanine (the spectroscopic peaks associated with alanine and lactate could not be reliably differentiated) were determined with MRS, and areas under the spectroscopic peaks (representing concentration estimates) were calculated. Ratios of these metabolite values were compared among age groups and among brain regions with regression analysis. RESULTS: The choline-to-creatine ratio was significantly higher in young dogs, compared with other age groups. The N-acetyl aspartate-to-choline ratio was significantly lower in young dogs and geriatric dogs than in adult dogs. When all age groups were considered, the choline-to-creatine ratio was significantly higher and N-acetyl aspartate-to-choline ratio was significantly lower in the frontal lobe than in all other regions. The N-acetyl aspartate-to-creatine ratio was significantly lower in the cerebellum than in other regions. CONCLUSIONS AND CLINICAL RELEVANCE: Metabolite ratios varied significantly among age groups and brain regions in healthy dogs. Future studies should evaluate absolute concentration differences in a larger number of dogs and assess clinical applications in dogs with neurologic diseases.

The effects of glycine on subjective daytime performance in partially sleep-restricted healthy volunteers.

Approximately 30% of the general population suffers from insomnia. Given that insomnia causes many problems, amelioration of the symptoms is crucial. Recently, we found that a non-essential amino acid, glycine subjectively and objectively improves sleep quality in humans who have difficulty sleeping. We evaluated the effects of glycine on daytime sleepiness, fatigue, and performances in sleep-restricted healthy subjects. Sleep was restricted to 25% less than the usual sleep time for three consecutive nights. Before bedtime, 3 g of glycine or placebo were ingested, sleepiness, and fatigue were evaluated using the visual analog scale (VAS) and a questionnaire, and performance were estimated by personal computer (PC) performance test program on the following day. In subjects given glycine, the VAS data showed a significant reduction in fatigue and a tendency toward reduced sleepiness. These observations were also found via the questionnaire, indicating that glycine improves daytime sleepiness and fatigue induced by acute sleep restriction. PC performance test revealed significant improvement in psychomotor vigilance test. We also measured plasma melatonin and the expression of circadian-modulated genes expression in the rat suprachiasmatic nucleus (SCN) to evaluate the effects of glycine on circadian rhythms. Glycine did not show significant effects on plasma melatonin concentrations during either the dark or light period. Moreover, the expression levels of clock genes such as Bmal1 and Per2 remained unchanged. However, we observed a glycine-induced increase in the neuropeptides arginine vasopressin and vasoactive intestinal polypeptide in the light period. Although no alterations in the circadian clock itself were observed, our results indicate that glycine modulated SCN function. Thus, glycine modulates certain neuropeptides in the SCN and this phenomenon may indirectly contribute to improving the occasional sleepiness and fatigue induced by sleep restriction.