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OBJECTIVES To characterise the clinical signs of suspected cerebrovascular disease in dogs. MATERIALS AND METHODS Medical records of one hospital were searched from November 2009 to December 2016 for dogs that suffered of cerebrovascular disease. We diagnosed cerebrovascular disease based on acute onset, clinical signs and magnetic resonance imaging findings. The medical history, clinical signs, concurrent disease, area of infarction, cerebrospinal fluid results, month at onset and outcome were investigated in the cerebrovascular disease group and in a control group (dogs with brain disorders other than cerebrovascular disease). RESULTS A total of 122 CVD cases were extracted from the 5312 patients that visited during the study period. Of these 122 cases, 66 (1.2%) matched the subject selection criteria of our study and were included in the analysis. Forebrain infarction was observed in 51 of 66 cases, of which 24 (47.1%) suffered from seizures. The number of dogs diagnosed with cerebrovascular disease was disproportionately high in August (nine of 59 cases) and December (13 of 59 cases). In the outcome survey, deterioration was observed in 11 of 55 cases. CLINICAL SIGNIFICANCE Seizure is an important clinical sign of cerebrovascular disease in dogs. There was a significant seasonal variation in the number of dogs diagnosed with cerebrovascular disease in Japan. Clinical features observed in this report differ from those of previous reports and highlight the need for additional research in this area.

Considering the importance of the canine cancer model of human disease, as well as the need for strategies for canine cancer management, the properties of exosomes are an emerging topic in canine oncology. In our study, exosomal RNA was isolated and investigated by next-generation sequencing. We identified several differentially expressed microRNAs (miRNAs/miRs) in the exosomes of two melanoma cell lines compared with non-tumor reference exosomes. We explored these potential melanoma-specific exosomal miRNAs further and found that miR-143 and let-7b increased in primary, whereas miR-210, 708, 221, and 222 increased in metastatic site originated melanoma cells. Further analysis showed miR-143 and 221 significantly increased in plasma exosomes of metastatic melanoma patients. Moreover, the sensitivity and specificity are >85% for differentiating the non-metastatic and metastatic patients. Therefore, these miRNAs can be an incredible biomarker candidate to identify metastatic melanoma and facilitate a better prognosis.

Background/aim The reproducibility of athero - sclerotic lesions was evaluated after the production of cloned-microminipigs and their offspring. Materials and methods Cloned-microminipig-parents were produced by microminipigsomatic cell nuclei. These parents were crossbred and delivered males (F1-offspring) were divided into two groups: normal chow diet (NcD)-fed and high-fat/high-cholesterol diet (HcD)-fed groups. One of the F1-offsprings was subjected to cloning, and delivered males (F1-clones) were fed with HcD. After 8 weeks, all animals were necropsied for patho - physiological studies compared to non-cloned-microminipigs. Results HcD-fed F1-offspring and F1-clones, but not NcD-fed F1-offspring, exhibited increased serum lipid levels and systemic atherosclerosis, which were comparable to those of HcD-fed non-cloned-microminipigs. Homogeneity of variance analysis demonstrated that standard deviation values of serum lipoprotein and aortic atherosclerosis area from HcD-fed animals decreased in F1-offspring and F1-clones. Conclusion HcD-induced atherogenesis was highly reproducible in F1-offsprings and F1-clones, indicating that the atherosclerosis-prone genomic background was preserved in the cloned-microminipigs, which can be used for studies on human atherosclerosis and related diseases.

Background Toceranib phosphate (TOC) could be made widely available for treating tumors in dogs if evidence shows that TOC inhibits recurrence after surgery. Objectives To investigate how postoperative adjuvant treatment with TOC modulates the tumor microenvironment (TME), by assessing effects on angiogenic activity, tumor-infiltrating regulatory T cells (Tregs), and intratumoral hypoxia. Animals Ninety-two client-owned dogs were included: 28 with apocrine gland anal sac adenocarcinoma, 24 with small intestinal adenocarcinoma, 22 with lung adenocarcinoma, and 18 with renal cell carcinoma. Methods Retrospective, multicenter study comparing time to progression (TTP) between 42 dogs treated by surgery and TOC and 50 dogs treated by surgery alone. Differences were analyzed in the expression of vascular endothelial growth factor receptor-2 (VEGFR2) and the number of Foxp3+ Tregs and hypoxia-inducible factor (HIF)-1α+ cells in tumor tissues sampled at the first and second (recurrence) surgeries. Results Median TTP for dogs treated by surgery and TOC (360 days) was higher than that for dogs treated by surgery alone (298 days; hazard ratio, 0.82; 95% confidence interval [CI], 0.65-0.96; P = .02). In dogs treated by surgery and TOC, VEGFR2 expression and the number of Tregs and HIF-1α+ cells were significantly lower in tissues sampled at the second surgery than in those sampled after the first surgery. In dogs treated by surgery alone, significant differences were found between samples from the 2 surgeries. Conclusions and clinical importance Toceranib phosphate could prove to be a useful postoperative adjuvant treatment because of its modulation of the TME.

The expression levels of circulating microRNAs (miRNAs) can be affected by disease. The miRNA released from cells within exosomes can act as a remote communication tool and can participate in inflammatory response regulation. Therefore, circulating miRNA has the potential to be an indicator of local disease. The objective of this study was to investigate the serum level of bovine mastitis-related miRNAs. We found that miR-16 expression in serum was affected by hemolysis. The expression levels of miR-21 in serum were increased significantly in cows with mastitis compared with unaffected controls; however, the expression levels of miR-146a, miR-155, miR-222 and miR-383 in cows with mastitis were unchanged. We further verified the upregulation of miR-21 in the serum of cows with mastitis using a digital PCR system. Although the sensitivity and specificity of miR-21 in the serum to detect bovine mastitis was inferior to miRNA biomarkers in the milk, the significant increase of miR-21 in serum may reflect the impact of local inflammation on the systemic reaction.

Critical thinking (CT) is important for consensus building. Although the practice of CT using debate is widely used to improve open-minded thought, the cognitive processes underlying this improvement remain poorly understood. Here, functional magnetic resonance imaging (fMRI) was employed to assess how neural responses while considering another's opinion are changed by CT practice and to determine whether the cortical network showing activation changes related to personality traits is relevant to consensus building. A total of 52 healthy participants were divided into three groups for an intervention; the participants read another's reasoning regarding a controversial issue and judged whether this person's viewpoint was affirmative during fMRI measurements. The intervention required them to prepare speech texts from a designated viewpoint based on both themselves and others. Compared to the control group, the group who took another's perspective showed enhanced activation of the right parieto-frontal network, that has been implicated in belief update, cognitive reappraisal of emotion, and self-perspective inhibition. Additionally, activation of the orbitofrontal cortex was negatively correlated with a stubbornness index. The results provide the first neural evidence of the effects of CT practice and support the notion that open-minded thought underlies the benefits of this practice.

Mastitis is a common inflammatory infectious disease in dairy cows. To understand the microRNA (miRNA) expression profile changes during bovine mastitis, we undertook a genome-wide miRNA study of normal milk and milk that tested positive on the California mastitis test for bovine mastitis (CMT+). Twenty-five miRNAs were differentially expressed (23 miRNAs upregulated and two downregulated) during bovine mastitis relative to their expression in normal milk. Upregulated mature miR-1246 probably derived from a U2 small nuclear RNA rather than an miR-1246 precursor. The significantly upregulated miRNA precursors and RNU2 were significantly enriched on bovine chromosome 19, which is homologous to human chromosome 17. A gene ontology analysis of the putative mRNA targets of the significantly upregulated miRNAs showed that these miRNAs were involved in binding target mRNA transcripts and regulating target gene expression, and a Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the upregulated miRNAs were predominantly related to cancer and immune system pathways. Three novel miRNAs were associated with bovine mastitis and were relatively highly expressed in milk. We confirmed that one of the novel mastitis-related miRNAs was significantly upregulated using a digital PCR system. The differentially expressed miRNAs were involved in human cancers, infections, and immune-related diseases. The genome-wide analysis of miRNA profiles in this study provides insight into bovine mastitis and inflammatory diseases. DATABASES: The miRNAseq generated for this study can be found in the Sequence Read Archive (SRA) under BioProject Number PRJNA421075 and SRA Study Number SRP126134 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA421075).

Dogs have been considered as an excellent immunocompetent model for human melanoma due to the same tumor location and the common clinical and pathological features with human melanoma. However, the differences in the melanoma transcriptome between the two species have not been yet fully determined. Considering the role of oncogenes in melanoma development, in this study, we first characterized the transcriptome in canine oral melanoma and then compared the transcriptome with that of human melanoma. The global transcriptome from 8 canine oral melanoma samples and 3 healthy oral tissues were compared by RNA‑Seq followed by RT‑qPCR validation. The results revealed 2,555 annotated differentially expressed genes, as well as 364 novel differentially expressed genes. Dog chromosomes 1 and 9 were enriched with downregulated and upregulated genes, respectively. Along with 10 significant transcription site binding motifs; the NF‑κB and ATF1 binding motifs were the most significant and 4 significant unknown motifs were indentified among the upregulated differentially expressed genes. Moreover, it was found that canine oral melanoma shared >80% significant oncogenes (upregulated genes) with human melanoma, and JAK‑STAT was the most common significant pathway between the species. The results identified a 429 gene signature in melanoma, which was up‑regulated in both species; these genes may be good candidates for therapeutic development. Furthermore, this study demonstrates that as regards oncogene expression, human melanoma contains an oncogene group that bears similarities with dog oral melanoma, which supports the use of dogs as a model for the development of novel therapeutics and experimental trials before human application.

Purpose: Nonconventional vapor products (NVP), designed to reduce exposure to cigarette smoke toxicants (CSTs), could cause changes in biomarkers of potential harm (BoPH). Although, NVPs re...

Porcine epidemic diarrhea virus (PEDV) induces an often fatal gastrointestinal disease in piglets. In this study, we performed a PEDV infection experiment with the Microminipig, the smallest of experimental minipigs, as a novel small animal model. We orally inoculated a neonatal Microminipig with an intestinal homogenate of a PEDV-infected pig and housed it in a small cage originally designed for rats in an animal biosafety level 2 facility. The infected Microminipig showed the typical signs of porcine epidemic diarrhea (PED), such as watery diarrhea, loss of appetite and weight loss. We also recognized a high amount of excreted PEDV in its rectal swabs and villus atrophy of the small intestine. These results suggest that the Microminipig is a good small animal model for PED, which may contribute to a better understanding of the pathogenesis of PEDV.

The changes in intra-atrial blood coagulability of acute phase after development of atrial fibrillation (AF) have not been elucidated in human. In the present study, blood coagulability were examined in the intra-atrial and peripheral regions during the acute phase after development of rapid atrial pacing (RAP) in experimentally created model dog similar to AF, using Total Thrombus-formation Analysis System (T-TAS) that is capable of comprehensively evaluating thrombogenicity in the bloodstream in the microvascular channel. According to the results, both the coagulating function-evaluating time to +10 kPa (T10) and occlusion time (OT) of the AR chip (chip for thrombus analysis mixed with coagulation and platelet) were significantly shortened in the atrial blood as early as 30 min after pacing (T10, 150.5 ± 40.5 s; OT, 212.4 ± 44.3 s) compared to the pre-pacing levels (T10, 194.5 ± 47.5 s; OT, 259.9 ± 49.5 s) (P

Phenylalanine and serine are amino acids used in dietary supplements and nutritional products consumed by healthy consumers, however, the safe level of phenylalanine or serine supplementation is unknown. The objective of this study was to conduct two 4-week clinical trials to evaluate the safety and tolerability of graded dosages of oral phenylalanine and oral serine. Healthy male adults (n = 60, 38.2 ± 1.8y) completed graded dosages of either phenylalanine or serine supplement (3, 6, 9 and 12 g/d) for 4 weeks with 2-week wash-out periods in between. Primary outcomes included vitals, a broad spectrum of circulating biochemical analytes, body weight, sleep quality and mental self-assessment. At low dosages, minor changes in serum electrolytes and plasma non-essential amino acids glutamine and aspartic acid concentrations were observed. Serine increased its plasma concentrations at high supplemental dosages (9 and 12 g/day), and phenylalanine increased plasma tyrosine concentrations at 12 g/day, but those changes were not considered toxicologically relevant. No other changes in measured parameters were observed, and study subjects tolerated 4-week-long oral supplementation of phenylalanine or serine without treatment-related adverse events. A clinical, no-observed-adverse-effect-level (NOAEL) of phenylalanine and serine supplementation in healthy adult males was determined to be 12 g/day.

In dogs, pancreatic acinar cell injury is thought to be caused by decreased pancreatic blood flow due to heart failure. In previous our report, it demonstrated that decreased heart function causes a significant decrease in pancreatic blood flow in heart failure dog model caused by rapid ventricular pacing (RVP). However, the types of histopathological changes remain unclear. We aimed to verify the types of histopathological changes occurring in the pancreatic tissue due to decreased heart function. After RVP for 4 weeks, atrophy of pancreatic acinar cells, characterized by a decrease in zymogen granules, was observed in all areas of the pancreas. In conclusion, the result of this study suggests that attention should be paid to ischemia/hypoperfusion injury in the pancreas.

To date, coagulation tests are unable to reflect in vivo coagulation status in the same system, including platelet function, fibrin clot formation, and whole blood flow. The Total Thrombus Analysis System (T-TAS), which is a microfluidic assay that simulates conditions in vivo, measures whole blood flow at defined shear rates under conditions designed to assess platelet function (PL-chip) or coagulation and fibrin clot formation (AR-chip). The T-TAS records occlusion start time, occlusion time, and area under the curve. We evaluated this test in healthy control dogs. We also investigated the effect in vivo of acetylsalicylic acid (ASA), and the effect in vitro of an anticoagulation drug (dalteparin; low-molecular-weight heparin; LMWH). The CV of the AUC of both chips was good (CVs of 6.45% [PL] and 1.57% [AR]). The inhibition of platelet function by ASA was evident in the right-shift in the PL test pressure curve. The right-shift in the AR test pressure curves showed that the administration of LMWH inhibited both platelets and the coagulation cascade. The T-TAS may be useful in the evaluation of canine blood coagulation.

Nucleophosmin 1 (NPM1) primarily localizes to the nucleus and is passively released into the extracellular milieu by necrotic or damaged cells, or is secreted by monocytes and macrophages. Extracellular NPM1 acts as a potent inflammatory stimulator by promoting cytokine production [e.g., tumor necrosis factor-α (TNF-α)], which suggests that NPM1 acts as a damage-associated molecular pattern. However, the receptor of NPM1 is unknown. Evidence indicates that DAMPs, which include high mobility group box 1 and histones, may bind Toll-like receptors (TLRs). In the present study, it was shown that NPM1 signaling was mediated via the TLR4 pathway, which suggests that TLR4 is an NPM1 receptor. TLR4 binds myeloid differentiation protein-2 (MD-2), which is essential for intracellular signaling. Furthermore, the TLR4 antagonist, LPS-Rhodobacter sphaeroides (an MD-2 antagonist) and TAK-242 (a TLR4 signaling inhibitor) significantly inhibited NPM1-induced TNF-α production by differentiated THP-1 cells as well as reducing ERK1/2 activation. Far-western blot analysis revealed that NPM1 directly bound MD-2. Thus, the results of the present study provide compelling evidence that TLR4 binds NPM1, and it is hypothesized that inhibiting NPM1 activity may serve as a novel strategy for treating TLR4-related diseases.

MicroRNAs (miRNAs) can rapidly respond to cellular stresses, such as hypoxia. This immediate miRNA response regulates numerous genes and influences multiple signaling pathways. Therefore, identifying hypoxia-regulated miRNAs (HRMs) is important in canine oral melanoma (COM) to investigate their clinical significance. The hypoxic and normoxic miRNA profiles of two COM cell lines were investigated by next generation sequencing. HRMs were identified by comparing miRNA expression profiles in these cell lines with that in COM tissue. The HRM profile was different between cell lines of primary and metastatic origin, except for miR-301a and miR-8884. The time course of miRNA expression determined by qRT-PCR, especially for miR-210 and miR-301a, showed that metastatic cells are more resistant to hypoxia than primary cells. Analysis of an experimentally validated human miRNA target database revealed that miR-21 and miR-301a control a complex gene regulatory network in response to hypoxia, which includes pathways of well-known oncogenes, such as VEGF, PTEN, and TGFBR2. In conclusions, we revealed the HRM of COM. Moreover, our study shows the difference in regulation and response of hypoxic miRNAs between primary and metastatic originated melanoma cells.

Hemorrhagic diseases are common in dogs. Current coagulation assays do not model all aspects of in vivo hemostasis and may not predict bleeding risk. The Total-Thrombus Analysis System (T-TAS) is a novel hemostasis assay system in which whole blood flows through microfluidic channels at defined shear rates to provide qualitative and quantitative evaluation of platelet function (PL-chip) and coagulation function (AR-chip). The present study evaluated the T-TAS in dogs with hereditary bleeding disorders and with acquired hemorrhagic syndromes (Group 1), and healthy controls (Group 2). Hereditary defects included von Willebrand's disease (VWD; n = 4), hemophilia A (n = 2), and canine Scott syndrome (n = 2). Acquired hemorrhagic disorders included neoplastic hemoperitoneum (n = 2) and acute hemorrhagic diarrhea syndrome (n = 1). Citrate anticoagulated samples were collected from diseased dogs (Group 1, n = 11) and controls (Group 2, n = 11) for coagulation screening tests, fibrinogen analyses, D-dimer concentration, antithrombin activity, von Willebrand Factor antigen, PFA-100 closure time (PFA-CT), and thromboelastography (TEG). Citrate and hirudin anticoagulated samples were used for T-TAS analyses at two shear rates. Qualitative thrombus formation in each chip was recorded using the T-TAS video camera. Numeric parameters, derived from the instrument software, included occlusion start time (OST; time to 10 kPa), occlusion time (OT; time to 60 kPa (PL-chip) or 80 kPa (AR-chip)), and area under the pressure curve (AUC). Correlations between continuous variables were evaluated by Spearman's rank. Continuous variables were compared between groups by Student's t-test or the Mann-Whitney U-test. Alpha was set at 0.05. In combined analyses of all dogs, significant correlations were identified between T-TAS variables, between the PFA-CT and PL-chip parameters and between TEG variables and AR-chip parameters. The prothrombin time correlated with the AR-chip AUC at both shear rates. In Group 1 dogs, the AR-chip AUC at low shear was significantly reduced compared with Group 2 dogs. Aberrant thrombus formation was seen in video images recorded from dogs with VWD and hemophilia A. The T-TAS AR-chip analysis distinguished dogs with bleeding risk compared to healthy controls. Initial evaluations of the T-TAS suggest it may aid characterization of hemostasis in patients at-risk of bleeding and assist with delineating bleeding phenotypes.

The pancreas is believed to be vulnerable to hypoperfusion. In dogs with acute pancreatitis, pancreatic ischemia due to heart failure can worsen the condition. However, changes in pancreatic blood flow associated with decreased cardiac function have not been previously studied in dogs. Therefore, we aimed to identify and compare changes in pancreatic versus renal blood flow as a result of cardiac dysfunction. Seven dogs were subjected to rapid ventricular pacing to create heart failure models. Noninvasive blood pressure measurement, ultrasonic cardiography, contrast-enhanced ultrasonography for pancreatic blood flow measurement, and para-aminohippuric acid clearance for renal blood flow measurement were performed before starting and at 2 and 4 weeks after starting the pacing. Left ventricular cardiac output and mean blood pressure decreased at 2 and 4 weeks after starting the pacing, and pancreatic blood flow decreased at 2 and 4 weeks after starting the pacing. However, renal blood flow did not change at 2 weeks but decreased 4 weeks after starting the pacing. Overall, this study demonstrated that reduced pancreatic blood flow due to cardiac dysfunction occurs, similar to renal blood flow. This suggests that decreased pancreatic blood flow is not unusual and may frequently occur in dogs with heart failure. The results of this study support the speculation that heart failure can exacerbate acute pancreatitis. Additionally, this study provides useful basic information for designing further studies to study this association.

Background/aim An appropriate animal model is essential to investigate the relationship between inflammation, atherosclerosis, and thrombogenesis, and the development of preventive measures and therapies for atherosclerosis. Materials and methods Atherosclerosis was induced in Microminipigs (MMPs) using a high-fat diet. We assessed high mobility group box 1 (HMGB1) expression levels and measured thrombus formation using a Total Thrombus Formation Analysis System (T-TAS). MMPs were divided into a normal diet (control) group and four high-fat diet groups, with differing amounts of cholesterol. After 8 weeks, blood was collected for analysis. Results HMGB1 levels increased with increasing dietary cholesterol, and a negative correlation was found between HMGB1 levels and thrombus formation time. Conclusion T-TAS is useful in the assessment of thrombogenesis in MMPs and HMGB1 is associated with thrombus formation.

Background: There are various types of sleep disorders, such as insomnia, hypersomnia, and rhythm disorder, which are attributed to diverse and complex background factors. Recently, many studies have reported that lactic acid bacteria, bifidobacteria, and lactoferrin, are related to fatigue and sleep. Considering these factors, we prepared a test food samples containing lactic acid bacteria ingredients (fermentation products, living bacteria, and ground bacteria) derived from the lactic acid bacterium strain Lactobacillus helveticus (LBH_MIKI-020) and theanine, which is known to have a relaxing effect, and subjectively and objectively tested its effects on the quality of sleep. Methods: In this placebo-controlled, double-blinded, randomized crossover study, we randomly selected 40 male and female subjects (aged 20-64 years) to consume for four weeks test food (lactic acid bacteria ingredients –containing) tablets and placebo control food. The physical examination and laboratory test, sleep electroencephalography, Ogri-Shirakawa-Azumi sleep questionnaire (OSA sleep questionnaire), Pittsburgh Sleep Quality Index (PSQI), and Visual Analogue Scale (VAS) questionnaire were measured at during this study period. Results: Sleep electroencephalography: the intergroup comparison in changes until the 4th week showed a significant increase (improvements) compared with placebo control food group. In addition, the sleep efficiency (SE) was compared within the group. In the test food group, the SE increased (improved) significantly in 4 weeks. OSA sleep questionnaire: in intragroup analyses between week 0 and week 4, significant increases (improvements) were found in the test food group. PSQI: in intragroup comparisons between week 0 and week 4, significant decreases (improvements) were found in the test food group. VAS questionnaire: in intragroup comparisons between week 0 and week 4, significant decreases (improvements) were found in the test food group. Conclusion: The sleep efficiency (SE) is improved by continuous consumption of test food (lactic acid bacteria-containing food). Among various sleep disorders, a large population in Japan has trouble with the quality of sleep. Consumption of lactic acid bacteria-containing foods can be a useful means to safely improve the quality of sleep from the viewpoint of self-medication as well. Keywords: lactobacillus helveticus ; lactic acid bacteria; clinical trial; quality of sleep; sleep EEG; OSA sleep questionnaire; PSQI

Introduction Changes in blood characteristics in the atrium and peripheral vessels in patients with non-valvular atrial fibrillation (NVAF) are unclear. We investigated chronological changes in blood characteristics in the atrium and peripheral vessels in a dog model of NVAF by using a total thrombus-formation analysis system (T-TAS). Materials and methods In NVAF model dogs (n = 8, 390 bpm rapid atrial pacing), atrial and peripheral blood samples were collected. Using this blood, T-TAS was performed before and 1, 2, and 3 weeks after the onset of rapid atrial pacing. Results Occlusion time (OT: time to +80 and +60 kPa in the AR and PL chips, respectively) and area under the flow pressure curve (AUC) were measured using the AR chip (for mixed white thrombus analysis) and PL chip (for platelet thrombus analysis). OT of the AR chip showed shortening as early as 1 week after NVAF onset, which continued for 3 weeks. OT of the PL chip showed significant shortening in atrium blood only 3 weeks after NVAF onset. By contrast, peripheral blood showed no significant changes in OT or AUC with both AR and PL chips. Conclusions In our dog model of NVAF, thrombus formation accelerated in the atrium as early as 1 week after NVAF onset and continued for 3 weeks, but no significant changes were found in peripheral blood. We conclude that antithrombotic therapy should be started early after NVAF onset even if no changes in coagulation activity are observed in peripheral blood.

We examined whether multidrug resistant (MDR) canine lymphoma increases gene expression for platelet-derived growth factor receptor α (PDGFRα), vascular endothelial growth factor receptor 2 (VEGFR2), and c-KIT, and whether toceranib phosphate (TOC) has potential as a treatment for MDR canine lymphoma. The clinical data showed that PDGFRα expression was higher in canine subjects with MDR T-cell lymphoma than in those with untreated T-cell lymphoma, and that c-KIT expression was greater in subjects with T-cell lymphoma than in those with B-cell lymphoma. TOC monotherapy was well tolerated without serious adverse effects, and two of the five subjects that received TOC exhibited partial responses. The data presented here could contribute to the assessment of TOC-based therapy for dogs with MDR or T-cell lymphoma.

Our aim was to identify a suitable microRNA housekeeping gene for real-time PCR analysis of bovine mastitis-related microRNA in milk. We identified , , and as housekeeping gene candidates on the basis of previous Solexa sequencing results. Threshold cycle (CT) values for , , and did not differ between milk from control cows and milk from mastitis-affected cows. NormFinder software identified as the most stable single housekeeping gene. We evaluated the suitability of the housekeeping gene candidates by using them to assess expression levels of the inflammation-related gene . Regardless of the housekeeping gene candidates used for normalization, relative expression levels of were significantly higher in mastitis-affected samples than in control samples. However, of all the housekeeping genes and gene combinations investigated, normalization with alone generated the difference in relative expression between mastitis-affected and control samples with the highest significance. These results suggest that is suitable for use as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

The combination of alteplase, a recombinant tissue plasminogen activator, and edaravone, an antioxidant, reportedly enhances recanalization after acute ischemic stroke. We examined the influence of edaravone on the thrombolytic efficacy of alteplase by measuring thrombolysis using a newly developed microchip-based flow-chamber assay. Rat models of embolic cerebral ischemia were treated with either alteplase or alteplase-edaravone combination therapy. The combination therapy significantly reduced the infarct volume and improved neurological deficits. Human blood samples from healthy volunteers were exposed to edaravone, alteplase, or a combination of alteplase and edaravone or hydrogen peroxide. Whole blood was perfused over a collagen- and thromboplastin-coated microchip; capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 69.9% lower in the edaravone-alteplase- than alteplase-treated group. The thrombolytic effect of alteplase was significantly attenuated in the presence of hydrogen peroxide, suggesting that oxidative stress might hinder thrombolysis. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, edaravone significantly inhibited the decrease. Edaravone enhances alteplase-mediated thrombolysis, likely by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

Aim We have recently established a novel swine model for studies of atherosclerosis using MicrominipigsTM (µMPs) fed a high-fat/high-cholesterol diet (HcD). Using this swine model, we re-evaluated the effects of dietary cholic acid (CA) on serum lipid profile, atherosclerosis and hepatic injuries. Methods The µMPs were fed HcD supplemented with 0.7% CA (HcD＋CA) for eight weeks, and the effect of CA on serum lipoprotein levels, expression of oxidative stress markers, adiposity and lesion formation in the aorta, liver, and other organs was investigated. Results The HcD＋CA-fed group exhibited more visceral adiposity, progression of atherosclerosis and higher serum levels of oxidative stress markers than the HcD-fed group, even though they showed similar serum lipid levels. The liver demonstrated increased lipid accumulation, higher expression of oxidative stress markers, accelerated activation of foamy Kupffer cells and stellate cells, and increased hepatocyte apoptosis, indicating non-alcoholic fatty liver disease (NAFLD). Intriguingly, foamy macrophage mobilization was observed in various organs, including the reticuloendothelial system, pulmonary capillary vessels and skin very often in HcD＋CA-fed µMPs. Conclusion To our knowledge, this is the first large animal model, in which visceral obesity, NAFLD and atherosclerosis are concomitantly induced by dietary manipulation. These data suggest the detrimental effects of CA, potentially through local and systemic activation of oxidative stress-induced signaling to macrophage mobilization, on the acceleration of visceral adiposity, atherosclerosis and NAFLD.

MicroRNAs (miRNAs) dysregulation contribute the cancer pathogenesis. However, the miRNA profile of canine oral melanoma (COM), one of the frequent malignant melanoma in dogs is still unrevealed. The aim of this study is to reveal the miRNA profile in canine oral melanoma. MiRNAs profile of oral tissues from normal healthy dogs and COM patients were compared by next-generation sequencing. Along with tumour suppressor miRNAs, we report 30 oncogenic miRNAs in COM. The expressions of miRNAs were further confirmed by quantitative real-time PCR (qPCR). Pathway analysis showed that deregulated miRNAs impact on cancer and signalling pathways. Three oncogenic miRNAs targets (miR-450b, 301a, and 223) from human study also were down-regulated in COM and had a significant negative correlation with their respective miRNA. Furthermore, we found that miR-450b expression is higher in metastatic cells and regulated MMP9 expression through a PAX9-BMP4-MMP9 axis. In silico analysis indicated that miR-126, miR-20b, and miR-106a regulated the highest numbers of differentially expressed transcription factors with respect to human melanoma. Chromosomal enrichment analysis revealed the X chromosome was enriched with oncogenic miRNAs. We comprehensively analyzed the miRNA&rsquo, s profile in COM which will be a useful resource for developing therapeutic interventions in both species.

While the human body maintains homeostasis by altering the balance in the autonomic nervous, endocrine, and immune systems, a prolonged imbalance in these systems can result in physical and mental symptoms, including a decline in sleep quality and work efficiency. Euglena gracilis (Euglena) is a single-celled microalga with the properties of both plants and animals and contains abundant nutrients, such as vitamins, minerals, amino acids, and fatty acids, which have various beneficial health effects. This study evaluated the effects of Euglena intake on the mood states and stress coping under mental workload tasks, and subjective sleep quality. We assigned men and women aged 20 to 64 years to Euglena and placebo intake groups, and measured indices related to the autonomic nervous system, psychological states, and sleep quality together with the application of workload stress before food intake, and 4, 8, and 12 weeks after commencing intake. Euglena intake regulated the autonomic nervous system under a workload and improved psychological parameters and sleep conditions. These results indicate that the consumption of Euglena may regulate the balance of the autonomic nervous system during stress and may have a favorable effect on psychological status and sleep quality.

Background Staphylococcus aureus is responsible for large numbers of hospital-related and community-acquired infections. In this study, we investigated the presence of S. aureus and methicillin-resistant S. aureus (MRSA) in 100 samples from animals (55 cattle, 36 dogs, and 9 cats) and 150 samples from hospitalized human patients. The samples were collected from healthy and diseased animals and from diseased humans and included milk, wound swab, pus, exudates, nasal swab and diabetic ulcer. Initially, S. aureus was isolated and identified by colony morphology, Gram staining, and biochemical tests (catalase and coagulase tests). The S. aureus-positive samples were examined by polymerase chain reaction (PCR) to determine their MRSA status. Results Of the 100 animal samples, 29 were positive for S. aureus. Four samples (13.8%) from dogs were MRSA-positive, but samples from cattle and cats were MRSA-negative. Of the 150 human samples we collected, 64 were S. aureus-positive and, of these, 34 (53.1%) were MRSA-positive. Most (28%) of the MRSA samples were isolated from surgical wound swabs, followed by the pus from skin infections (11%), exudates from diabetic ulcers (6%), exudates from burns (4%), and aural swabs (3%). By contrast, a low MRSA detection rate (n = 4) was seen in the non-human isolates, where all MRSA bacteria were isolated from nasal swabs from dogs. The antimicrobials susceptibility testing results showed that S. aureus isolates with mecA genes showed resistance to penicillin (100%), oxacillin (100%), erythromycin (73.5%), ciprofloxacin (70.6%), and gentamicin (67.7%). The lowest resistance was found against ceftazidime, and no vancomycin-resistant isolates were obtained. Conclusions We detected S. aureus and MRSA in both human and canine specimens. Isolates were found to be resistant to some of the antimicrobials available locally. MRSA carriage in humans and animals appears to be a great threat to effective antimicrobials treatment. The prudent use of antimicrobials will reduce the antimicrobial resistance. Our findings will help to find the most appropriate treatment and to reduce antimicrobial resistance in the future by implementing prudent use of antimicrobials. Further studies are required to better understand the epidemiology of MRSA human–animal inter-species transmission in Bangladesh. Electronic supplementary material The online version of this article (10.1186/s12917-018-1611-0) contains supplementary material, which is available to authorized users.

Previous studies reported that rendering the fixation point is effective for suppressing visually induced motion sickness (VIMS). This study investigates why the fixation point has a VIMS suppression effect. Gazing-specific-place and gazing-specific-object are two properties of static-fixation points. To clarify which property is responsible for the VIMS suppression effect of fixation points, we employ a moving-fixation point and employ 3D CG content experienced by participants through a head-mounted display (HMD). This study compares the following: no-fixation-point, static-fixation-point, and reciprocating-fixation-point conditions. Each condition is evaluated by a simulator sickness questionnaire (SSQ) and subjective evaluations. Both the SSQ and subjective evaluation results show the VIMS suppression effect and compare it under the reciprocating-fixation-point condition and with the no-fixation-point condition. The gazing-specific-object property might cause a VIMS suppression effect.

Background/aim The aim of this study was to develop a method for sequentially collecting cerebrospinal fluid (CSF) from an unanesthetized microminipig, which shares many physiological and anatomical similarities with humans, such as diurnality, and investigate the diurnal variation of melatonin concentration in the CSF. Materials and methods A catheter was placed percutaneously into the subarachnoid space of an anesthetized animal, and the tip of the catheter was placed into the cisterna magna under X-ray. We then sequentially collected CSF at light-on and -off times from the unanesthetized animal for several weeks. After catheter placement, a period of one week or more was necessary to relieve the contamination of RBCs in the CSF. Results A higher melatonin level in the CSF was noted during lights-off time, and the level was higher than that in the serum. Conclusion This model of sequential collection of CSF will contribute to research in brain functions.

This study aimed to evaluate the usefulness of near-infrared time-resolved spectroscopy (TRS) for the monitoring of post-resuscitation encephalopathy. Cardiac arrest (CA) was induced in pigs by electrical stimuli; then, return of spontaneous circulation (ROSC) was achieved by direct current. The changes in cerebral oxygenation were analyzed by two methods: (1) the time-independent calculation based on the modified Beer-Lambert law (MBL), and (2) the curve-fitting method based on the photon diffusion theory (DT). The changes in reduced scattering coefficient (μs′) in DT were also calculated. Post-resuscitation encephalopathy was evaluated by MRI findings. During CA, cerebral oxygen saturation (ScO2) decreased to the lowest level, and then gradually increased during the chest compression period. When ROSC was achieved, ScO2 (DT) increased further, but ScO2 (MBL) decreased transiently. This strange phenomenon disappeared when the scalp was peeled off and the probes were directly fixed to the cranial bone. In some cases, a sustained decrease in μs′ was observed several hours after ROSC and, in such cases, MRI Diffusion Enhancement Image (DWI) showed findings suggestive of post-resuscitation encephalopathy. In conclusion, simultaneous monitoring of cerebral oxygenation with MBL and DT may provide more information about the vascular response of different layers. Also, the monitoring of μs′ may help us to recognize the occurrence of post-resuscitation encephalopathy in real time.

Canine hepatocellular carcinoma (HCC) is the most common primary hepatic tumour in dogs. MicroRNA (miRNA) dysregulation has been reported in human HCC and shown to have diagnostic and prognostic value; however, there are no data on miRNA expression in canine HCC. The aim of the present study was to investigate differentially expressed miRNAs in canine HCC. Analysis of miRNA expression in canine HCC tissues and cell lines by quantitative reverse transcription PCR showed that miR-1, miR-122, let-7a, and let-7g were downregulated, whereas miR-10b and miR-21 were upregulated in canine HCC. MET is one of the target genes of miR-1. MET was upregulated in canine HCC at the gene and protein levels, and a significant correlation between the concomitant downregulation of miR-1 and upregulation of MET was observed. Fast/intermediate-proliferating canine HCC cell lines had higher MET gene and protein expression levels than the slow-proliferating cell line. These findings suggest that miRNAs are differentially expressed in canine HCC, and that the miR-1/MET pathway may be associated with canine HCC cell proliferation.

Angiotensin (Ang) II is known to promote vascular disease and hypertension, partly through its effect on vascular endothelium. Bradykinin (BK) is an endothelium-dependent agonist that induces relaxation followed by contraction of the porcine basilar artery through release of NO and PGF2α, respectively. In this study, we evaluated the effect of Ang II-induced hypertension on basilar artery responsiveness to BK in the Microminipig (MMPig). Ang II (200ng/kg/min) or vehicle was infused into MMPigs for 14days using an osmotic mini-pump and blood pressure was monitored regularly. The responsiveness of subsequently isolated basilar arteries was then measured using a micro organ bath system. MMPig basilar artery endothelial cells were cultured and stimulated with Ang II or vehicle for 48h. Mean blood pressure was significantly (P

MicroRNA (miRNA) in tissue and liquid samples have been shown to be associated with many diseases including inflammation. We aimed to identify inflammation-related miRNA expression level in the bovine mastitis milk. Expression level of inflammation-related miRNA in milk from mastitis-affected and normal cows was analyzed using qPCR. We found that expression level of miR-21, miR-146a, miR-155, miR-222, and miR-383 was significantly upregulated in California mastitis test positive (CMT+) milk. We further analyzed these miRNA using a chip-based QuantStudio Digital PCR System. The digital PCR results correlated with those of qPCR, demonstrating upregulation of miR-21, miR-146a, miR-155, miR-222, and miR-383 in CMT+ milk. In conclusion, we identified miRNA that are upregulated in CMT+ milk. These miRNA exhibited sensitivity and specificity greater than 80% for differentiating between CMT+ milk and normal milk. Our findings suggest that inflammation-related miRNA expression level in the bovine milk was affected by mastitis, and miRNA in milk have potential for use as biomarkers of bovine mastitis.

Cartilage regeneration with cell therapy following arthroscopic surgery could be used in racehorses with intra-articular fractures (IAF) and osteochondritis dissecans (OCD). The aims of this study were to investigate the origin and multipotency of stromal cells in the synovial fluid (SF) of horses with intra-articular injury and synovitis, and to provide a new strategy for regeneration of lost articular cartilage. Mesenchymal stromal cells were isolated from SF of horses with IAF and OCD. Multipotency was analysed by RT-PCR for specific mRNAs and staining for production of specific extracellular matrices after induction of differentiation. The total number of SF-derived mesenchymal stromal cells reached >1 × 107 by the fourth passage. SF-derived cells were strongly positive (>90% cells positive) for CD44, CD90 and major histocompatibility complex (MHC) class I, and moderately positive (60–80% cells positive) for CD11a/CD18, CD105 and MHC class II by flow cytometry. SF-derived cells were negative for CD34 and CD45. Under specific nutrient conditions, SF-derived cells differentiated into osteogenic, chondrogenic, adipogenic and tenogenic lineages, as indicated by the expression of specific marker genes and by the production of specific extracellular matrices. Chondrogenic induction in culture resulted in a change in cell shape to a ‘stone-wall’ appearance and formation of a gelatinous sheet that was intensely stained with Alcian blue. SF may be a novel source of multipotent mesenchymal stem cells with the ability to regenerate chondrocytes.

Bradyarrhythmia is usually a spontaneously subsiding complication of cervical spinal cord injury. However, in severe cases, it can lead to cardiac arrest. We report a case of cervical spinal cord injury, complicated by right-sided infective endocarditis after the placement of a temporary pacing catheter in the right ventricle for severe bradyarrhythmia that led to cardiac arrest. Although the patient׳s condition was successfully treated by pacing catheter removal and pharmacological therapy, right-sided infective endocarditis would be a fatal complication in cases of cervical spinal cord injury where cardiac pacing is required for long-term refractory severe bradyarrhythmia.

A 10-month-old Japanese black heifer was diagnosed as having an intra-abdominal cyst using computed tomography (CT). Through a posterior ventral midline incision, the cyst was removed, and the heifer completely recovered after the surgery. CT scans enabled detection of the intra-abdominal cyst and measurements of the diameter of the cyst before the surgery.

This study investigated the relationship between plasma and saliva cotinine kinetics after smoking one cigarette and the relationship between cotinine kinetics and estimated nicotine intake, which was calculated as mouth level exposure (MLE) of nicotine, from smoking two test cigarettes with different nicotine yields. This study was conducted in 16 healthy adult Japanese smokers, who did not have null nor reduced-activity alleles of CYP2A6, with a quasi-randomized crossover design of smoking a low-tar cigarette or a high-tar cigarette. Saliva cotinine showed similar concentration profiles to plasma cotinine, and all of the calculated pharmacokinetic parameters of cotinine showed the same values in plasma and saliva. The Cmax and AUC of cotinine showed almost the same dose-responsiveness to the estimated MLE of nicotine between plasma and saliva, but the tmax and t1/2 of cotinine were not affected by the estimated MLE of nicotine in either plasma or saliva. The results show that saliva cotinine kinetics reflects plasma cotinine kinetics, and measurement of saliva cotinine concentration gives the same information as plasma cotinine on the nicotine intake. Thus, saliva cotinine would be a good and less-invasive exposure marker of cigarette smoke, reflecting the plasma cotinine concentration and kinetics.

SummaryHigh residual platelet aggregability and circulating platelet-monocyte aggregates in patients administered aspirin and clopidogrel are associated with ischaemic vascular events. To determine the relevance of these factors with residual thrombogenicity, we measured platelet thrombus formation using a microchip-based flow-chamber system in cardiac patients receiving aspirin and/or clopidogrel, and evaluated its correlation with agonist-inducible platelet aggregation and platelet-monocyte aggregates. Platelet thrombus formation was analysed by measuring flow pressure changes due to the occlusion of micro-capillaries and was quantified by calculating AUC10 (area under the flow pressure curve). The growth and stability of platelet thrombi that formed inside microchips at shear rates of 1000, 1500, and 2000 s-1 were markedly reduced in patients receiving aspirin and/or thienopyridine compared to healthy controls (n=33). AUC10 values of aspirin therapy patients (n=20) were significantly lower and higher than those of healthy controls and dual antiplatelet therapy patients (n=19), respectively, and showed relatively good correlations with collagen-induced platelet aggregation and platelet-monocyte aggregates at 1000 and 1500 s-1 (r s >0.59, p0.59, p

The intrinsic value of an action refers to the inherent sense that experiencing a behavior is enjoyable even if it has no explicit outcome. Previous research has suggested that a common valuation mechanism within the reward network may be responsible for processing the intrinsic value of achieving both the outcome and external rewards. However, how the intrinsic value of action is neurally represented remains unknown. We hypothesized that the intrinsic value of action is determined by an action-outcome contingency indicating the behavior is controllable and that the outcome of the action can be evaluated by this feedback. Consequently, the reward network should be activated, reflecting the generation of the intrinsic value of action. To test this hypothesis, we conducted a functional magnetic resonance imaging (fMRI) investigation of a stopwatch game in which the action-outcome contingency was manipulated. This experiment involved 36 healthy volunteers and four versions of a stopwatch game that manipulated controllability (the feeling that participants were controlling the stopwatch themselves) and outcome (a signal allowing participants to see the result of their action). A free-choice experiment was administered after the fMRI to explore preference levels for each game. The results showed that the stopwatch game with the action-outcome contingency evoked a greater degree of enjoyment because the participants chose this condition over those that lacked such a contingency. The ventral striatum and midbrain were activated only when action-outcome contingency was present. Thus, the intrinsic value of action was represented by an increase in ventral striatal and midbrain activation.

Purpose The assessment of retinal nerve fiber layer defects (RNFLDs) is a useful part of glaucoma care. Here, we obtained en-face images of retinal layers below the inner limiting membrane (ILM) with swept source-optical coherence tomography (SS-OCT), and measured RNFLD angle with new software. Methods This study included 105 eyes of 105 normal tension glaucoma (NTG) patients (age, 59.8 ± 13.2). Exclusion criteria were best-corrected visual acuity 28 mm, non-glaucoma ocular disease, and systemic disease affecting the visual field. We obtained 12 x 9 mm 3D volume scans centered on the macula with SS-OCT (DRI OCT-1, Topcon), and from these scans, created 3 averaged en-face images, each comprising 7 horizontal en-face images (total thickness: 18.2 μm). We labeled these averaged images, according to their depth below the ILM, as en-face images 1 (shallowest), 2 (middle) and 3 (deepest). In each image, a circle was drawn centered on the disc, with a radius of half the distance between the centers of the disc and macula. The investigator marked points where the edge of the RNFLD intersected this circle, and RNFLD angle (RNFLDA) was calculated with new software. Finally, we analyzed the association between RNFLDA, cpRNFLT, weighted RGC count (wrgc) and Humphrey field analyzer (HFA)-measured mean deviation (MD) and hemifield total deviation (TD), both overall and in each hemifield. Results En-face image 2 had the highest interclass reproducibility for measuring RNFLDA (intra-rater intraclass correlation coefficient (ICC): 0.988, inter-rater ICC: 0.962). The correlation coefficients with RNFLDA were: HFA MD, -0.60; superior TD, -0.73; inferior TD, -0.69; overall cpRNFLT, -0.27; superior hemifield cpRNFLT, -0.39; and inferior hemifield cpRNFLT, -0.53 (all p

Introduction Despite the proven benefits of thrombolytic therapy with tissue plasminogen activator (t-PA) for peripheral thromboembolism, perfusion failure frequently occurs, particularly in arterial circulation. We evaluated how the modification of fibrinolytic activity affects thrombus formation under flow and static conditions. Materials and methods t-PA-treated human whole-blood samples (n = 6) were perfused over a microchip coated with collagen and tissue thromboplastin at different shear rates, and thrombus formation was quantified by measuring flow pressure changes. For comparison, rotational thromboelastometry (ROTEM) was used to evaluate fibrinolytic activity under static conditions. Results At a shear rate of 240 s − 1 , t-PA (200–800 IU/ml) concentration-dependently delayed capillary occlusion, whereas at 600 s − 1 , capillary occlusion was significantly faster and t-PA had limited effects, even at a supra-pharmacological concentration (800 IU/ml). In contrast, 200 IU/ml t-PA efficiently prevented clot formation in the ROTEM assay. The combined treatment of blood with a specific PAI-1 inhibitor (PAI-039) moderately enhanced the efficacy of t-PA, but only under flow conditions. In addition, 1:1-diluted blood samples of PAI-1-deficient (−/−) mice showed a significant delay of capillary occlusion at 240 s − 1 , compared with those from wild-type mice (1.55 fold; P Conclusions The present results suggest that the anti-thrombotic efficacy of t-PA is sensitive to arterial shear flow, and that PAI-1 secreted from activated platelets plays an essential role in thrombolytic resistance.