Characterization of the oral and faecal microbiota associated with atopic dermatitis in dogs selected from a purebred Shiba Inu colony.

Atopic dermatitis (AD) is a chronic and relapsing multifactorial inflammatory skin disease that also affects dogs. The oral and gut microbiota are associated with many disorders, including allergy. Few studies have addressed the oral and gut microbiota in dogs, although the skin microbiota has been studied relatively well in these animals. Here, we studied the AD-associated oral and gut microbiota in 16 healthy and 9 AD dogs from a purebred Shiba Inu colony. We found that the diversity of the oral microbiota was significantly different among the dogs, whereas no significant difference was observed in the gut microbiota. Moreover, a differential abundance analysis detected the Family_XIII_AD3011_group (Anaerovoracaceae) in the gut microbiota of AD dogs; however, no bacterial taxa were detected in the oral microbiota. Third, the comparison of the microbial co-occurrence patterns between AD and healthy dogs identified differential networks in which the bacteria in the oral microbiota that were most strongly associated with AD were related to human periodontitis, whereas those in the gut microbiota were related to dysbiosis and gut inflammation. These results suggest that AD can alter the oral and gut microbiota in dogs.

Three-dimensional SAFT imaging for anisotropic materials using photoacoustic microscopy.

A pulsed laser illuminates a target zone that causes rapid thermoelastic expansion, generating broadband high-frequency ultrasonic wave (photoacoustic wave, PA wave). We developed a PA microscopy (PAM) with a confocal area of laser and ultrasonic wave for applications in nondestructive testing (NDT). The synthetic aperture focusing technique (SAFT) is applied in the PAM for the three-dimensional (3D) imaging of interior flaws. Here, we report proof-of-concept experiments for the NDT of a subsurface flaw in a thin laminar material. Graphical abstract (a) shows a specimen of carbon-fiber-reinforced plastic (CFRP) with an artificial delamination. Here, it should be noted that the group velocity varies directionally due to the strong anisotropy of the CFRP specimen (see Graphical abstract (b)). By considering the group velocity distribution in the SAFT, the shape and location of the subsurface delamination were accurately estimated as shown in Graphical abstract (c). Coating the surface of the CFRP specimen with a light-absorbent material improved the amplitude of the PA wave. This finding showed that the signal-to-noise ratio of the waves scattered from the flaws can be improved.

Survey of Two New (Kai 1 and Kai 2) and Other Blood Groups in Dogs of North America.

BACKGROUND: Based upon serology, >10 canine blood group systems have been reported. OBJECTIVE: We surveyed dogs for dog erythrocyte antigen (DEA) 1 and 2 new blood types (Kai 1 and Kai 2), and some samples also were screened for Dal and DEA 3, 4, and 7. METHODS: Blood samples provided by owners, breeders, animal blood banks, and clinical laboratories were typed for DEA 1 by an immunochromatographic strip technique with a monoclonal antibody and analysis of band intensity. Both new antigens, the Dal and other DEAs (except DEA 7 by tube method), were assessed by a gel column method with either monoclonal or polyclonal antibodies. The same gel column method was applied for alloantibody detection. RESULTS: Of 503 dogs typed, 59.6% were DEA 1+ with 4% weakly, 10% moderately, and 45.6% strongly DEA 1+. Regarding Kai 1 and Kai 2, 94% were Kai 1+/Kai 2-, 5% were Kai 1-/Kai 2- and 1% were Kai 1-/Kai 2+, but none were Kai 1+/Kai 2+. There was no relationship between Kai 1/Kai 2 and other blood types tested. Plasma from DEA 1-, Kai 1-, Kai 2- dogs, or some combination of these contained no detectable alloantibodies against DEA 1 and Kai 1 or Kai, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: The new blood types, called Kai 1 and Kai 2, are unrelated to DEA 1, 3, 4, and 7 and Dal. Kai 1+/Kai 2- dogs were most commonly found in North America. The clinical relevance of Kai 1 and Kai 2 in canine transfusion medicine still needs to be elucidated.

Molecular prevalence of multiple genetic disorders in Border collies in Japan and recommendations for genetic counselling.

Reproductive management is necessary to prevent deleterious genetic disorders in purebred dogs, but comprehensive studies aimed at prevention of multiple underlying genetic disorders in a single breed have not been performed. The aims of this study were to examine mutant allele frequencies associated with multiple genetic disorders, using Border collies as a representative breed, and to make recommendations for prevention of the disorders. Genotyping of known mutations associated with seven recessive genetic disorders was performed using PCR assays. More than half (56%) of the Border collies had no mutant alleles associated with any of the seven disorders, suggesting that these disorders can be removed from the population over several generations. Since frequencies of each mutant allele differed among disorders, reproductive management should be performed after the establishment of prevention schemes that are appropriate for each disorder, the type and specificity of genetic test available, and the effective population size in each breeding colony.

Prototyping the Experimental Setup to Quantify the Tissue Oxygen Consumption Rate and Its Preliminary Test.

In order to establish a reliable and practical method to make a diagnosis on the viability of an amputated extremity, we propose a method to evaluate the oxygen consumption rate. To validate this concept, we prototyped an experimental system with which the oxygen transfer rate into tissue can be assessed by the rate of change of the decrease in dissolved oxygen (DO) concentration within the buffer fluid surrounding the target tissue. The purpose of this study is to examine the feasibility of our prototyped experimental system by comparison between fresh and non-fresh rat skeletal muscles. The results show that the fresher tissue transferred more oxygen to the tissue, which suggests that tissue oxygen consumption is highly related to tissue freshness and can indirectly assess the tissue viability.

Expression of Autophagy-Related Proteins in the Spinal Cord of Pembroke Welsh Corgi Dogs With Canine Degenerative Myelopathy.

Canine degenerative myelopathy (DM) is a progressive neurodegenerative disease frequently found in Pembroke Welsh Corgi (PWC) dogs, and it has clinical and pathologic similarities to human amyotrophic lateral sclerosis. Autophagy is a major intracellular protein degradation system. Abnormalities of autophagy--resulting in cell death through mechanisms called type II programmed cell death--have recently been reported to occur in various neurodegenerative diseases, including amyotrophic lateral sclerosis. Thus, the distribution and expression levels of proteins involved in autophagy were examined in the spinal cords of 8 PWC dogs suffering from DM with superoxide dismutase mutation, 5 non-DM PWC dogs, and 6 Beagle dogs without neurologic signs. There was no significant difference in the ratio of neurons with microtubule-associated protein light chain 3 (LC3)-positive somata relative to those that were LC3 negative among the 3 groups, whereas the number of LC3-positive neurites was significantly increased in DM dogs. Punctate LC3 immunoreactivity did not colocalize with a lysosome marker, LAMP2 (lysosome-associated membrane protein 2). NBR1 (neighbor of BRCA gene 1) was localized mostly in reactive astrocytes, whereas there were p62 (p62/A170/SQSTM1)-positive foci in the neuropil of the spinal cord of DM dogs. Western blotting revealed in DM dogs the decreased expression of Beclin1 and Atg16 L, which are molecules involved in formation of the isolation membrane. These findings suggest that altered autophagosome degradation may result in LC3 and p62 accumulation in the DM spinal cord, whereas the early stage of membrane formation is likely to be downregulated.

Feline cystinuria caused by a missense mutation in the SLC3A1 gene.

BACKGROUND: Cystinuria is an inherited metabolic disease that is relatively common in dogs, but rare in cats and is characterized by defective amino acid reabsorption, leading to cystine urolithiasis. OBJECTIVES: The aim of this study was to report on a mutation in a cystinuric cat. ANIMALS: A male domestic shorthair (DSH) cat with cystine calculi, 11 control cats from Wyoming, and 54 DSH and purebred control cats from elsewhere in the United States. METHODS: Exons of the SLC3A1 gene were sequenced from genomic DNA of the cystinuric cat and a healthy cat. Genetic screening for the discovered polymorphisms was conducted on all cats. RESULTS: A DSH cat showed stranguria beginning at 2 months of age, and cystine calculi were removed at 4 months of age. The cat was euthanized at 6 months of age because of neurological signs possibly related to arginine deficiency. Twenty-five SLC3A1 polymorphisms were observed in the sequenced cats when compared to the feline reference sequence. The cystinuric cat was homozygous for 5 exonic and 8 noncoding SLC3A1 polymorphisms, and 1 of them was a unique missense mutation (c.1342C>T). This mutation results in a deleterious amino acid substitution (p.Arg448Trp) of a highly conserved arginine residue in the rBAT protein encoded by the SLC3A1 gene. This mutation was found previously in cystinuric human patients, but was not seen in any other tested cats. CONCLUSIONS AND CLINICAL IMPORTANCE: This study is the first report of an SLC3A1 mutation causing cystinuria in a cat, and could be used to characterize other cystinuric cats at the molecular level.

Mutational analysis of the feline CLN3 gene and an ultrastructural evaluation of lysosomal storage materials in a cat with neuronal ceroid lipofuscinosis: an investigation into the molecular basis of the disease.

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease caused by a number of different genes. A mutational analysis of the feline CLN3 gene was performed in a cat with NCL that had vacuolated lymphocytes, which is a feature of human NCL caused by defects of the CLN3 gene. To determine the candidate gene(s) responsible for this case, NCL-specific ultrastructures of storage materials were analysed. A sequence analysis indicated that the CLN3 gene was not likely to be responsible for this case of feline NCL because no deleterious mutation was detected. An ultrastructural analysis did not reveal any candidate gene because of inconsistency with any pattern found in human NCL. These findings suggest that the diagnostic criteria for human NCL are not directly applicable to feline NCL.

Vascular calcification and secondary hyperparathyroidism of severe chronic kidney disease and its relation to serum phosphate and calcium levels.

BACKGROUND AND PURPOSE: Various complications consequent on disordered calcium and phosphate homeostasis occur frequently in chronic kidney disease (CKD) patients. Particularly, vascular calcification has high morbidity and mortality rates. There is a clear need for a better CKD model to examine various aspects of this disordered homeostasis. EXPERIMENTAL APPROACH: Oral dosing with adenine induced CKD in rats in only 10 days. Serum calcium, phosphate and parathyroid hormone were measured and calcification in aorta was assessed histologically. The effects of varying phosphorus content of diet or treatment with phosphate binders or active vitamin D(3) on these parameters were examined. KEY RESULTS: After adenine dosing, significant hyperphosphatemia, hypocalcemia and secondary hyperparathyroidism (2HPT) were observed during the experimental period of 15 weeks. Aortic calcification was detected in only some of the animals even at 15 weeks (approximately 40%). Treatment with vitamin D(3) for 18 days, even at a low dose (100 ng x kg(-1), 3-4 times week(-1), p.o), caused aortic calcification in all animals and increases in serum calcium levels up to the normal range. The vitamin D(3)-induced calcification was significantly inhibited by phosphate binders which lowered serum phosphate levels and the calcium x phosphate product, although serum calcium levels were elevated. CONCLUSIONS: These data suggest that rats dosed orally with adenine provide a more useful model for analysing calcium/phosphate homeostasis in severe CKD. Controlling serum calcium/phosphate levels with phosphate binders may be better than vitamin D(3) treatment in hyperphosphatemia and 2HPT, to avoid vascular calcification.

CLOCK gene T3111C polymorphism is associated with Japanese schizophrenics: a preliminary study.

The CLOCK gene has attracted attention due to its influence on the circadian rhythm, as well as its impacts on the dopaminergic system. We conducted a preliminary study to examine whether the T3111C single nucleotide polymorphism of the CLOCK gene is associated with the development of schizophrenia by examining samples from schizophrenics (n=145) and normal controls (n=128). Both genotype and allele frequencies were significantly different between schizophrenics and controls (p=0.022, p=0.015, respectively). Schizophrenics had a significantly higher frequency of the C allele compared to controls (odds ratio 1.76, 95% CI 1.12-2.75). In particular, disorganized and residual type schizophrenics had significantly higher C allele frequencies than controls (p=0.004 and p=0.037, respectively). Our results suggest that the T3111C polymorphism of the CLOCK gene is associated with schizophrenia. It is important to explore the association between CLOCK and dopamine function, and to examine the impact of CLOCK on phenotypes such as symptoms and drug response in patients with schizophrenia.

The Eating Disorder Inventory-2 in Japanese clinical and non-clinical samples: psychometric properties and cross-cultural implications.

OBJECTIVE: To determine the usefulness of and cross-cultural differences in the Japanese version of the eating disorder inventory-2 (EDI-2). METHOD: A Japanese version of the EDI-2 was administered to a consecutive series (1995-2001) of 91 Japanese eating disordered inpatients and 119 matched non-clinical controls. Scores were compared with each other and with those of Canadian data previously reported. Internal consistencies were also examined. RESULTS: All subscales (except Ascetism) reached sufficient internal consistencies. The patients' mean scores were significantly higher than controls on most subscales. Restricting anorexics (AN-R) scored higher than controls on body dissatisfaction (BD) and Social Insecurity (SI) subscales. Purging bulimics (BN-P) scored higher than controls on most subscales, except for maturity fears (MF) and SI subscales. Scores of binge-eating/purging type anorexics (AN-B/P) were higher than controls on all subscales. AN-B/P and BN-P scored higher than AN-R on bulimia (B) subscale. Comparison with Canadian subjects showed that Japanese control subjects scored significantly higher on the MF, ineffectiveness (I), impulse regulation (IR), SI subscales, and lower on the perfectionism (P) subscale. DISCUSSION: The EDI-2 is useful and reliable in understanding Japanese clinical and non-clinical samples. High MF and low P subscales in our samples confirm previous findings in Chinese samples suggesting meaningful cultural differences in the emphasis placed on family bonds and individual accomplishments. In addition, high I, IR, and SI scores in Japanese non-clinical samples may reflect recent problems in Japanese culture.

Alterations of ubiquitin immunoreactivity in the hippocampal formation after perforant pathway lesion.

Immunohistochemical techniques were employed to examine the changes in free ubiquitin within the hippocampus 1, 3, 7, 14, and 30 days after a unilateral perforant pathway lesion occurred in the rat brain. Immunoreactivity for ubiquitin was remarkably decreased in the cell body and proximal dendrites of neurons throughout the hippocampus ipsilateral to the lesion at 1 day post-lesion. At 3 days post-lesion, ubiquitin immunoreactivity was recovered in interneurons in the whole hippocampus as well as in mossy cells in the hilar region, although granule cells in the dentate gyrus and pyramidal cells in the CA1 subfield remained unlabeled, and pyramidal cells in the CA3 subfield demonstrated only weak immunoreactivity. In addition, we observed an increase in ubiquitin immunolabeling of the hilar neuropil ipsilateral to the lesion at 1 and 3 days post-lesion, and a decrease in immunolabeling in the inner portion of the molecular layer at 3 days post-lesion. All these alterations were transient, and by 7 days post-lesion, ubiquitin immunoreactivity was indistinguishable in the hippocampus ipsilateral to the lesion, compared to the controls. Immunoblot analysis also revealed a decrease in the amount of ubiquitin in the hippocampus ipsilateral to the lesion 1 and 3 days post-lesion. These data suggest that deafferentation of the perforant pathway results in transient reduction in free ubiquitin of the hippocampus, and that the ubiquitin system is involved in hippocampal plasticity following perforant lesions.

Role of endogenous PACAP in catecholamine secretion from the rat adrenal gland.

We elucidated the contribution of endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) to neurally evoked catecholamine secretion from the isolated perfused rat adrenal gland. Infusion of PACAP (100 nM) increased adrenal epinephrine and norepinephrine output. The PACAP-induced catecholamine output responses were inhibited by the PACAP type I receptor antagonist PACAP- (6-38) (30-3,000 nM) but were resistant to the PACAP type II receptor antagonist [Lys1,Pro2,5,Ara3,4,Tyr6]-vasoactive intestinal peptide (LPAT-VIP; 30-3,000 nM). Transmural electrical stimulation (ES; 1-10 Hz) or infusion of ACh (6-200 nM) increased adrenal epinephrine and norepinephrine output. PACAP-(6-38) (3,000 nM), but not LPAT-VIP, also inhibited the ES-induced catecholamine output responses. However, PACAP-(6-38) did not affect the ACh-induced catecholamine output responses. PACAP at low concentrations (0.3-3 nM), which had no influence on catecholamine output, enhanced the ACh-induced catecholamine output responses, but not the ES-induced catecholamine output responses. These results suggest that PACAP is released from the nerve endings to facilitate the neurally evoked catecholamine secretion through PACAP type I receptors in the rat adrenal gland.

NAD/NADH models with axial/central chiralities: superiority of the quinoline ring system.

Precursors of NAD model compounds 1c and 3a,b were successfully resolved into their atropisomers with respect to carbamoyl rotation. Atropisomers of quinoline derivatives are much more stable than pyridine derivatives as determined by cyclic voltammetry and X-ray crystallography. The 1,4-reduction of NAD model compound 4 was successfully achieved, affording novel NADH model compound 5. The rotational properties of the side chain of 5 were investigated by means of dynamic NMR. The rotational rate and syn/anti ratio, which indicate the orientation between carbonyl oxygen and hydrogen at the 4-position, are significantly affected by addition of magnesium ion. In the rotational transition state, the double-bond character of the C(carbonyl)-N(amide) bond is disrupted judging from the activation parameters. The oxidation of chiral 5 with p-benzoquinone in the presence of magnesium ion catalyst gave predominantly one enantiomer of 4. On the other hand, oxidation of 5 with p-chloranil (tetrachloro-p-benzoquinone) in the absence of magnesium ions affords the opposite enantiomer of 4 as the major product. The product enantiomer ratio is parallel to the syn/anti ratio in the starting material, indicating the importance of ground state conformation to stereochemistry of the reaction.

Age-related loss of the AMPA receptor subunits GluR2/3 in the human nucleus basalis of Meynert.

Magnocellular cholinergic neurons in the basal forebrain have long been recognized as vulnerable to the pathology of Alzheimer's disease. Despite numerous anatomical, pharmacological, behavioral, and physiological investigations of these neurons the cellular mechanism that underlines their selective vulnerability remains unclear. As part of an ongoing investigation into the molecular mechanism(s) underlying neuronal vulnerability in Alzheimer's disease and normal aging, we employed immunocytochemical techniques and examined the cellular localization of the alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) glutamate receptor subunits GluR1 and GluR2/3 in the basal forebrain of eight nondemented elderly human subjects (66-102 years). For each case we observed GluR1-positive magnocellular cells darkly labeled within all main divisions of the basal forebrain (Ch1-Ch4). Double-labeling immunohistochemical techniques confirmed that the overwhelming majority (94%) of these neurons were also positive for the p75NGFr antibody, thus substantiating the cholinergic nature of these neurons. In contrast, GluR2/3 immunolabeling upon magnocellular neurons was relatively faint or nonexistent. The latter observations were most apparent in cases of advanced age and in the posterior part of the nucleus basalis of Meynert (NBM) (i.e., Ch4). In contrast, in adjacent structures (e.g., globus pallidus), a number of robustly labeled GluR2/3-positive cells were observed. In addition to the eight elderly subjects, we examined GluR1 and GluR2/3 immunostaining in the NBM of five younger cases, 5, 33, 36, 47, and 48 years of age. Although practical considerations limited our observations to the Ch4 region, we observed both GluR1 and GluR2/3 labeling upon NBM neurons in this latter region. On average, the distribution of labeled cells and intensity of immunoreaction were comparable between GluR1 and GluR2/3. The presence of GluR2/3- and GluR1-labeled neurons in the Ch4 region of younger cases but primarily GluR1 in cases of advanced age suggests an age-related decrease in GluR2/3. Functionally, the loss of GluR2 from the AMPA receptor complex results in ion channels highly permeable to Ca(2+). These alterations in cation permeability of the AMPA receptor together with the occurrence of a number of other intrinsic and extrinsic events (i.e., decrease Ca(2+)-binding protein) likely contribute to the vulnerability of these neurons in aging and in AD.

Immunohistochemical study of the hnRNP A2 and B1 in the rat forebrain.

Immunohistochemical techniques were employed to examine the distribution of RNA-binding proteins A2 and B1 in the rat forebrain. Intense A2 and B1 immunolabeling were observed in the nucleoplasm of the neurons in the cerebral cortices, hippocampal formation, olfactory regions, caudate-putamen as well as the supraoptic nucleus of hypothalamus. In contrast, within the bed nucleus of the stria terminalis, as well as the medial and lateral habenular nucleus of thalamus, immunoreactivity for both proteins was weak. Within the globus pallidus and thalamic nucleus immunoreactivity for A2 was hardly detectable despite of intense B1 immunolabeling, while within the endopiriform nucleus and lateral and basolateral nucleus of amygdala intensity of B1 immunolabeling was relatively weak compared to A2. Our study suggests that the distribution of A2 and B1 are not constant throughout the forebrain and this diversity may reflect the post-transcriptional regulation of cell-specific gene expression of neuronal cells.