Elevated expression of miR-301a and its functional roles in canine oral melanoma.

miR-301a is one of numerous dysregulated microRNAs (miRNAs) in canine oral melanoma (COM), one of which is miR-301a (upregulated). Its biological role has been described in various human cancer types, including malignant melanoma, but not in COM. Accordingly, in this study, we investigated miR-301a expression in COM in greater detail to ascertain whether it could serve as a diagnostic biomarker, elucidate its functional roles in this cancer, and predict the possible pathways by which it exerts its effects. Relative expression of miR-301a was investigated in clinical oral tissue and plasma samples and COM cell (KMeC and LMeC) lines using qRT-PCR. Knockdown of miR-301a was also validated for KMeC and LMeC cells using qRT-PCR. We performed CCK-8 assays to assess cell proliferation, monolayer wound-healing, and transwell migration assays to assess cell migration, a colony-formation assay to assess clonogenicity, a TUNEL assay and flow cytometry to assess apoptosis-related effects, and gene enrichment analyses to predict possible related pathways. miR-301a was markedly upregulated in COM oral tissue and plasma clinically, suggesting its potential as a diagnostic biomarker for COM diagnosis. In vitro assays demonstrated that miR-301 significantly inhibited apoptosis in COM cells while promoting cell migration, proliferation, and clonogenicity. We also predicted that miR-301 exerts cancer-promoting effects through the Wnt signalling pathway for COM. Our findings suggest that miR-301a is a COM oncomiR that regulates several oncogenic phenotypes with the potential to be a diagnostic biomarker.

Fragment-Based Discovery of Novel VE-PTP Inhibitors Using Orthogonal Biophysical Techniques.

Tyrosine phosphorylation is an essential post-translational modification that regulates various biological events and is implicated in many diseases including cancer and atherosclerosis. Vascular endothelial protein tyrosine phosphatase (VE-PTP), which plays an important role in vascular homeostasis and angiogenesis, is therefore an attractive drug target for these diseases. However, there are still no drugs targeting PTP including VE-PTP. In this paper, we report the discovery of a novel VE-PTP inhibitor, Cpd-2, by fragment-based screening combining various biophysical techniques. Cpd-2 is the first VE-PTP inhibitor with a weakly acidic structure and high selectivity, unlike known strongly acidic inhibitors. We believe that this compound represents a new possibility for the development of bioavailable VE-PTP inhibitors.

Common bile duct perforation due to choledocholithiasis in a cat with gallbladder agenesis.

Case summary: An 8-year-old neutered male domestic shorthair cat was presented for further investigation of anorexia, vomiting and lethargy. Abdominal ultrasonography and contrast-enhanced CT revealed choledocholithiasis with suspected bacterial peritonitis and non-visualisation of the gallbladder. During surgery, the common bile duct was noted to be perforated, and a cholelith was found in the abdominal cavity. No gallbladder was confirmed during surgery. Three months postoperatively, the cat underwent CT cholangiography and absence of the gallbladder with a vestigial duplicated gallbladder was diagnosed. Relevance and novel information: Gallbladder agenesis is extremely rare in cats, with only one previous report, but several dogs have been diagnosed based on CT cholangiography and laparoscopy. This report describes gallbladder agenesis concurrent with choledocholithiasis in an adult cat and represents the first report of CT cholangiography in a cat with gallbladder agenesis.

Protective Effect on Pancreatic Acinar Cell by Maintaining Cardiac Output in Canine Heart Failure Model With Decreased Pancreatic Blood Flow.

Heart failure cause hypoperfusion-induced damage to abdominal organs due to decreased cardiac output (CO). Using a model dog with heart failure caused by rapid ventricular pacing (RVP), we have previously demonstrated that a decrease in CO reduces pancreatic blood flow (PBF). Furthermore, we have revealed that pancreatic acinar cell atrophy, which is a change in the pre-stage of pancreatitis was caused. However, the mechanism by which pancreatic acinar cell atrophy was caused in RVP dogs remains unknown. This study aimed to clarify the association between cardiac function, PBF, and histopathological changes in pancreatic acinar cells by administrating pimobendan, which increase CO, to RVP dogs. RVP dogs were divided into the control group (no medication, n = 5) and the pimobendan group (pimobendan at 0.25 mg/kg BID, n = 5). Non-invasive blood pressure measurement, echocardiography, and contrast-enhanced ultrasonography for PBF measurement were performed before initiating RVP and at 4 weeks after initiating RVP (4 weeks). At 4 weeks, the decreases in CO, mean blood pressure and PBF due to RVP were suppressed in pimobendan group. Furthermore, histopathological examination showed no changes in pancreatic acinar cells in the pimobendan group. Overall, it was clarified that the decrease in PBF due to cardiac dysfunction was a direct cause of pancreatic acinar cell atrophy. This suggests that maintaining PBF is clinically important for treating dogs with heart failure. In addition, these findings offer a reliable basis for developing new therapeutic strategies for heart failure in dogs, that is, pancreatic protection.

Structure-based drug design of novel and highly potent pyruvate dehydrogenase kinase inhibitors.

Pyruvate dehydrogenase kinases (PDHKs) are fascinating drug targets for numerous diseases, including diabetes and cancers. In this report, we describe the result of our structure-based drug design from tricyclic lead compounds that led to the discovery of highly potent PDHK2 and PDHK4 dual inhibitors in enzymatic assay. The C3-position of the tricyclic core was explored, and the PDHK2 X-ray structure with a representative compound revealed a novel ATP lid conformation in which the phenyl ring of Phe326 mediated the interaction of the Arg258 sidechain and the compound. Compounds with amide linkers were designed to release the ATP lid by forming an intramolecular pi-pi interaction, and these compounds showed single-digit nM IC50 values in an enzymatic assay. We also explored the C4-position of the tricyclic core to reproduce the interaction observed with the C3-position substitution, and the pyrrolidine compound showed the same level of IC50 values. By optimizing an interaction with the Asn255 sidechain through a docking simulation, compounds with 2-carboxy pyrrole moiety also showed single-digit nM IC50 values without having a cation-pi interaction with the Arg258 sidechain.

Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases.

A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 provided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions, the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water molecule in the pocket led to the identification of lead compound 7 with a notable improvement in inhibition potency. This lead compound possessed high ligand efficiency (LE) and showed decent selectivity profile. Two additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for the development of PDHKs inhibitors.

Histopathological changes in the pancreas due to decreased pancreatic blood flow in a canine tachycardia-induced cardiomyopathy model.

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.

Analysis of blood clotting with the total thrombus analysis system in healthy dogs.

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.

A Novel Microchip Flow Chamber (Total Thrombus Analysis System) to Assess Canine Hemostasis.

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.

Systemic Inonotus sp. Infection in a dog.

A 3-years-old male golden retriever was presented for decreased activity (lethargy), anorexia, and titubation. Superficial lymph nodes were enlarged, and arrhythmia and tachycardia were auscultated. Fungal hyphae-like structures were detected in the biopsy samples from an enlarged lymph node and spleen. Nucleotide sequence of the internal transcribed spacer region of the fungi amplified by PCR was highly homologous to that of Inonotus pachyphloeus. The dog was treated with antifungal agents such as itraconazole, fluconazole, and voriconazole. Clinical signs resolved for 325 days but the dog died suddenly, possibly because of arrhythmia. Postmortem examination revealed the presence of a disseminated fungal infection. This report describes the case of canine systemic Inonotus sp. infection treated by an antifungal agent.

Comparison of pancreatic and renal blood flow in a canine tachycardia-induced cardiomyopathy model.

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.

Identification of dysregulated microRNAs in canine malignant melanoma.

Inhibiting aberrantly upregulated microRNAs (miR/miRNAs) has emerged as a novel focus for therapeutic intervention in human melanoma. Thus, identifying upregulated miRNAs is essential for identifying additional melanoma-associated therapeutic targets. In the present study, microarray-based miRNA profiling of canine malignant melanoma (CMM) tissue obtained from the oral cavity was performed and differential expression was confirmed by a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). An analysis of the microarray data revealed 17 dysregulated miRNAs; 5 were upregulated and 12 were downregulated. RT-qPCR analysis was performed for 2 upregulated (miR-204 and miR-383), 3 downregulated (miR-122, miR-143 and miR-205) and 6 additional oncogenic miRNAs (oncomiRs; miR-16, miR-21, miR-29b, miR-92a, miR-125b and miR-222). The expression levels of seven of the miRNAs, miR-16, miR-21, miR-29b, miR-122, miR-125b, miR-204 and miR-383 were significantly upregulated; however, the expression of miR-205 was downregulated in CMM tissues compared with normal oral tissues. The microarray and RT-qPCR analyses validated the upregulation of two potential oncomiRs miR-204 and miR-383. The present study additionally constructed a protein interaction network and a miRNA-target regulatory interaction network using STRING and Cytoscape. In the proposed network, cyclin dependent kinase 2 was a target for miR-383, sirtuin 1 and tumor protein p53 were targets for miR-204 and ATR serine/threonine kinase was a target for both. It was concluded that miR-383 and miR-204 were potential oncomiRs that may be involved in regulating melanoma development by evading DNA repair and apoptosis.

Comparison between blood coagulability in the intra-atrial and peripheral regions during the acute phase after rapid atrial pacing.

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<0.05). The OT of PL chip (chip for platelet thrombus analysis) was significantly shortened 30 min after pacing (231.8 ± 57.6 s), compared to the pre-pacing level (289.5 ± 96.0 s) (P<0.05). Meanwhile, none of T10 and OT of AR and PL chips showed any significant changes in the peripheral blood. The study demonstrated increase of blood coagulability 30 min after development of RAP. While no significant changes were observed in the peripheral blood in the present study, the outcome suggested that the anti-thrombus treatments are better to be started early after AF even if coagulability of the peripheral blood shows no change.

Comparison of chronological changes in blood characteristics in the atrium and peripheral vessels after the development of non-valvular atrial fibrillation.

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.

Acquired Fanconi syndrome in two dogs following long-term consumption of pet jerky treats in Japan: case report.

Renal Fanconi syndrome has recently been associated with the ingestion of pet jerky treats from China in mostly small breed dogs in North America, Australia and Europe. We report here about two dogs with Fanconi syndrome following pet jerky treats exposure in Japan. A mixed-breed dog and a French bulldog showed weight loss, polyuria and polydipsia. For years, the owners had been feeding large quantities of pet jerky treats containing chicken prepared in China. Diagnostics revealed glycosuria without hyperglycemia, severe aminoaciduria, and in one case also ketonuria, hypokalemia and metabolic acidosis. A diagnosis of Fanconi syndrome associated with long-term consumption of Chinese pet jerky treats was made. Both dogs recovered fully following withdrawal of the pet jerky treats and supportive care. Fanconi syndrome of dogs in association with the consumption of pet jerky treats of Chinese origin can cause a broad proximal tubular defect with glycosuria and generalized amino aciduria, and should be also considered in Asia. Jerky treats associated Fanconi syndrome can be completely reversible following withdrawal of the treats and supportive care to correct the metabolic abnormalities.

Ganglion cysts arising from a canine stifle joint.

A 10-year-old, neutered male Labrador retriever presented with progressive left hind lameness. Ultrasonography revealed large, subcutaneous, ovoid cysts around the stifle joint. Radiographic and computed tomographic images revealed periosteal reaction of the distal femur. Magnetic resonance (MR) imaging showed a large cyst that was hypointense in T1-weighted images, hyperintense in T2-weighted images and had a thin lining that was enhanced by intravenous gadonium injection. The cyst communicated with the joint cavity and other small cysts around the joint. Histopathology of an excisional biopsy specimen led to diagnosis of ganglion cyst. This report provides MR images of a ganglion cyst in a canine stifle.

Abnormal erythroid cell proliferation and myelofibrosis in a cat.

A cat was presented with severe progressive anemia despite marked erythroblastosis. The cat was negative for feline leukemia virus antigen and feline immunodeficiency virus antibody. Bone marrow cytology revealed an excess of erythroid cells with a predominance of prorubricytes and basophilic rubricytes. No response to immunosuppressive therapy was obtained, and a tentative diagnosis of myelodysplastic syndrome was made. The cat showed a partial response to low-dose cytarabine (20 mg/m(2) subcutaneously q24) but died 51 days after the 1st admission. Histopathological examination revealed fibrosis in the bone marrow and marked infiltration of erythroid cells into other organs.

CT and MRI imaging diagnosis of epidural idiopathic sterile pyogranulomatous inflammation in a dog spinal canal.

A 12-year-old neutered male shih tzu developed progressive pelvic limb paraparesis. Computed tomography showed a radiolucent mass lesion in the spinal canal at the left side of the 11th thoracic vertebra. The mass was not enhanced by intravenous contrast medium injection. It was hyperintense on both T1- and T2-weighted magnetic resonance images. The signal intensity of the mass was decreased with a fat suppression technique, indicating a fatty origin. After removal of the mass via T11-T12 hemilaminectomy, chronic panniculitis was confirmed by histopathological examination. This case demonstrates the utility of computed tomography and magnetic resonance imaging for the diagnosis of spinal canal pyogranulomatous inflammation.