Upregulation and functional roles of miR-450b in canine oral melanoma.

Canine oral melanoma (COM) is a common and highly aggressive disease with the potential to model human melanomas. Dysregulated microRNAs represent an interesting line of research for COM because they are implicated in tumor progression. One example is miR-450b, which has been investigated for its molecular mechanisms and biological functions in multiple human cancers, but not human or canine melanoma. Here, we aimed to investigate miR-450b as a potential diagnostic biomarker of COM and its functional roles in metastatic and non-metastatic forms of the disease. We investigated the expression of miR-450b and its target mRNA genes in clinical (tumor tissue and plasma) samples and metastatic and primary-tumor cell lines. Knockdown and overexpression experiments were performed to determine the influence of miR-450b on cell proliferation, migration, colony formation, and apoptosis. miR-450b was significantly upregulated in COM and differentiated between metastatic and non-metastatic tumors, and its potential as a biomarker of metastatic and non-metastatic COM was further confirmed in ROC analysis. miR-450b knockdown promoted cell proliferation, migration, and clonogenicity and inhibited apoptosis, whereas its overexpression yielded the reverse pattern. miR-450b directly binds 3' UTR of PAX9 mRNA and modulates its function leading to BMP4 downregulation and MMP9 upregulation at the transcript level. Furthermore, we surmised that miR-450b activates the Wnt signaling pathway based on gene ontology and enrichment analyses. We concluded that miR-450b has the potential as a diagnostic biomarker and could be a target candidate for COM treatment.

Hypoxia-related Y RNA fragments as a novel potential biomarker for distinguishing metastatic oral melanoma from non-metastatic oral melanoma in dogs.

Hypoxia may promote tumor progression, and hypoxically altered noncoding RNA (ncRNA) expression may play a role in metastasis. Canine oral melanoma (COM) frequently metastasizes, and ncRNA expression under hypoxia may be clinically significant. We aimed to elucidate ncRNA fragments whose expression is altered by hypoxia in COM-derived primary KMeC and metastatic LMeC cell lines using next-generation sequencing to validate these results in qRT-PCR, and then compare expression between metastatic and non-metastatic COM. The NGS analysis and subsequent qRT-PCR validation were performed using hypoxic and normoxic KMeC and LMeC cells, and clinical samples [tumor tissue, plasma, and plasma-derived extracellular vesicles] obtained from dogs with metastatic or non-metastatic melanoma were analyzed with qRT-PCR. Y RNA was significantly decreased in metastatic LMeC cells versus primary KMeC cells in hypoxic and normoxic conditions. The expression of Y RNA was decreased in dogs with metastatic melanoma versus those with non-metastatic melanoma for all clinical sample types, reflecting the pattern found with hypoxia. Receiver operating characteristic analysis demonstrated that Y RNA level is a promising biomarker for discriminating metastatic from non-metastatic melanoma in plasma [area under the curve (AUC) = 0.993, p < 0.0001] and plasma-derived extracellular vesicles (AUC = 0.981, p = 0.0002). Overall, Y RNA may be more resistant to hypoxic stress in the metastatic than the non-metastatic state for COM. However, further investigation is required to elucidate the biological functions of Y RNA under hypoxic conditions.

YRNA and tRNA fragments can differentiate benign from malignant canine mammary gland tumors.

Mammary gland tumors (MGT) are the most common tumors in sexually intact female dogs. The functional regulation of miRNAs, a type of noncoding RNAs (ncRNAs), in canine MGT has been extensively investigated. However, the expression of other ncRNAs, such as YRNAs and transfer RNA-derived fragments (tRFs) in canine MGT is unknown. We investigated ncRNAs other than miRNAs from our small RNA project (PRJNA716131) in different canine MGT histologic subtypes. This study included benign tumors (benign mixed tumor, complex adenoma) and malignant tumors (carcinoma in benign tumor and carcinoma with metastasis) samples. Aberrantly expressed ncRNAs were examined by comparisons among MGT subtypes. The relative expression trends were validated in canine MGT tissues, plasma, extracellular vesicles, and MGT cell lines using quantitative reverse transcription PCR. Three aberrantly expressed ncRNAs were identified by comparisons among MGT subtypes. YRNA and tRNA-Gly-GCC distinguished benign mixed tumor from other MGT histologic subtypes, while tRNA-Val differentiated complex adenoma, carcinoma in benign tumors, and carcinoma with metastasis. The ROC curve of the three ncRNAs showed they might be potential biomarkers to discriminate malignant from benign MGT. YRNA and tRFs expression levels were decreased in metastatic compared with primary canine MGT cell lines. To the best of our knowledge, this is the first investigation of YRNA and tRFs in canine MGT. The three identified ncRNAs may be biomarkers for differentiating MGT histologic subtypes. Suggested Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporatio.

Development of a Microminipig Model of Atherosclerosis for the Evaluation of a HMGCR Inhibitor.

BACKGROUND/AIM: Atherosclerosis is known as a major risk factor for cardiovascular disease, and development of an animal model of atherosclerosis is required to investigate its clinical pathogenesis. We studied the optimal amount of cholesterol in the diet and the optimal experimental period for development of a Microminipig model of atherosclerosis for the evaluation of a hydroxymethylglutaryl-CoA reductase (HMGCR) inhibitor (atorvastatin). MATERIALS AND METHODS: Eighteen male animals (3-4 months old) were divided into 3 groups. Group 1 consisted of control animals receiving a normal chow diet, Group 2 animals received a high fat (12% w/w) and low cholesterol (0.1% w/w) diet (HFLCD), and Group 3 animals received HFLCD+statin for 12 weeks. Animals received statin at 3 mg/kg body weight per day. HFLCD did not down-regulate the hepatic expression of HMGCR mRNA. RESULTS: HFLCD increased body, omentum, and mesenteric adipose tissue weight, and induced hypercholesterolemia and atherosclerotic lesions in the abdominal aorta. HFLCD+statin inhibited hypercholesterolemia and atherosclerotic lesions, but not obesity. CONCLUSION: A microminipig atherosclerosis model induced by HFLCD can be used in the evaluation of HMGCR inhibitors for the treatment of hypercholesterolemia and atherosclerosis.

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.

Novel Y RNA-Derived Fragments Can Differentiate Canine Hepatocellular Carcinoma from Hepatocellular Adenoma.

Hepatocellular carcinomas (HCC) are common tumors, whereas hepatocellular adenomas (HCA) are rare, benign tumors in dogs. The aberrant expression of noncoding RNAs (ncRNAs) plays a pivotal role in HCC tumorigenesis and progression. Among ncRNAs, micro RNAs have been widely researched in human HCC, but much less widely in canine HCC. However, Y RNA-derived fragments have yet to be investigated in canine HCC and HCA. This study targeted canine HCC and HCA patients. We used qRT-PCR to determine Y RNA expression in clinical tissues, plasma, and plasma extracellular vesicles, and two HCC cell lines (95-1044 and AZACH). Y RNA was significantly decreased in tissue, plasma, and plasma extracellular vesicles for canine HCC versus canine HCA and healthy controls. Y RNA was decreased in 95-1044 and AZACH cells versus normal liver tissue and in AZACH versus 95-1044 cells. In plasma samples, Y RNA levels were decreased in HCC versus HCA and Healthy controls and increased in HCA versus Healthy controls. Receiver operating characteristic analysis showed that Y RNA could be a promising biomarker for distinguishing HCC from HCA and healthy controls. Overall, the dysregulated expression of Y RNA can distinguish canine HCC from HCA. However, further research is necessary to elucidate the underlying Y RNA-related molecular mechanisms in hepatocellular neoplastic diseases. To the best of our knowledge, this is the first report on the relative expression of Y RNA in canine HCC and HCA.

Five major outcomes of digitalization: relevance of a survival personality type during COVID-19 pandemic.

The COVID-19 pandemic required people to adapt rapidly to the digital transformation of society for social survival, which highlighted the divide between those who can and cannot digitalize. Previous studies investigated factors promoting adaptation to digitalization; however, outcomes from adaptation to a digitalized society have not been sorted into a parsimonious model, even though there should be several multifaceted outcomes (e.g., usefulness, economic profit, and social outcome), each of which is promoted by different factors. If the effects of individual background factors can be revealed, including the technical-environment and survival-relevant personality in relation to each outcome, it would help in the creation of a society where more people play an active role by adapting to digitalization. This study aimed to construct such a model by identifying major outcomes gained in a digitalized society and investigating individual factors that contribute to the degree of gain of each of these outcomes. Five dimensions were identified by online surveys and factor analysis: Socialization (outcomes derived from new social connections created online), Space-time (freedom from time and space constraints), Economics (monetary outcome by using digital services), and Information (ease and amount of acquisition of information) were the positive outcomes, whereas Loneliness (feelings of not being able to keep up with digitization) was identified as a negative outcome. We determined that technical-environmental factors (e.g., familiarity with digital techniques and the amount of money that can be used for digitalization) facilitated gain in four positive outcomes. Notably, leadership and conscientiousness facilitated the Socialization gain while etiquette suppressed it. These factors' effects would reflect the importance of a personality trait prioritizing construction and maintenance of social relationships. This study implies that material outcomes (i.e., Space-time, Economics, and Information) are promoted by technical-environmental support, whereas social outcomes may additionally require motivation and a positive attitude for purposeful social engagement.

Successful Treatment of Central Nervous System Lymphoma with Combination Therapy of Nimustine and Prednisolone in Two Dogs.

Of intracranial tumors, primary central nervous system lymphoma (PCNSL) is rare in dogs. Herein, we describe our experience with two dogs (a 3-year-old intact female toy poodle and a 5-year-old spayed female toy poodle) that developed neurological symptoms. Magnetic resonance imaging (MRI) revealed intracranial disseminated lesions. Cerebrospinal fluid (CSF) examination revealed pleocytosis and B-cell monoclonal proliferation in both cases. PCNSL or secondary central nervous system lymphoma (SCNSL) was diagnosed on the basis of MRI findings and CSF examinations. Nimustine (ACNU) is a nitrosourea alkylating agent, a class of drugs that includes lomustine. Nimustine is mainly used to treat human intracranial neoplasia because of its high permeability across the blood-brain barrier. The dogs in this study were treated with combined chemotherapy comprising nimustine and prednisolone, which achieved complete or nearly complete remission of neurological symptoms and long-term survival (>2583 days and 1218 days), but with problematic adverse effects. We determined that the dose of nimustine for canine PCNSL or SCNSL with intravenous infusion was 25-30 mg/m2 every 3-4 weeks for a total of four times; however, the data were insufficient to determine the optimal regimen.

Effects of diet containing astaxanthin on visual function in healthy individuals: a randomized, double-blind, placebo-controlled, parallel study.

Visual display terminal work has increased rapidly in recent years. Loss of visual acuity is an unfortunate associated effect. Here, we performed a randomized, placebo-controlled study in 60 healthy adults. Participants received a diet containing astaxanthin (9 mg/day) or placebo for 6 weeks. Visual acuity, functional visual acuity, and pupil constriction rate were measured before and after visual display terminal work. In participants aged ≥40 years, corrected visual acuity of the dominant eye after visual display terminal work at 6 weeks after intake demonstrated a higher protective effect of astaxanthin in the astaxanthin group vs the control group (p<0.05). In participants aged <40 years, no significant difference was seen between the astaxanthin and control groups. Moreover, no significant difference was found in functional visual acuity and pupil constriction rate between the astaxanthin and control groups. These results suggest astaxanthin reduces oxidative stress caused by visual display terminal work. Age-related reduction in ciliary muscle strength is likely the main detractor of visual acuity. Correspondingly, astaxanthin reduced visual display terminal work-induced visual stress in the middle-aged and elderly. This study was registered in the UMIN-CTR database (UMIN000043089).

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.

microRNAs Are Abundant and Stable in Platelet-Rich Fibrin and Other Autologous Blood Products of Canines.

Various microRNAs (miRNAs) present in autologous blood products of canines have not been studied recently. We aimed to elucidate the existence of miRNAs in platelet-rich fibrin (PRF) and the stability of canine autologous blood products under various storage conditions. Total RNAs were isolated from PRF and other autologous blood products following newly adapted protocols used in commercial kits for plasma and tissue samples. Quantitative real-time polymerase chain reaction analysis (qPCR) was used to detect miRNAs in autologous blood products. The miR-16, miR-21, miR-155, and miR-146a were abundant in PRF and other autologous blood products of canines. Furthermore, we found they could maintain stability under protracted freezing temperatures of -30 °C for at least one month. Our findings revealed that PRF might be a stable resource for various canine miRNAs.

Subchronic tolerance trials of graded oral supplementation with ornithine hydrochloride or citrulline in healthy adults.

Ornithine and citrulline are amino acids used in dietary supplements and nutritional products consumed by healthy consumers, but the safe supplementation levels of these compounds are 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 ornithine (as hydrochloride) and citrulline. Healthy male adults (n = 60, age 41.4 ± 1.5 years) completed graded dosages of either ornithine hydrochloride (3.2, 6, 9.2, and 12 g/day) or citrulline (6, 12, 18, and 24 g/day) supplement 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. In the ornithine hydrochloride supplementation group, minor increase in plasma aspartic acid and glutamic acid concentrations was observed at the highest intake dosages. In the citrulline supplementation group, minor changes in laboratory data for serum lactate dehydrogenase and plasma amino acid concentration of lysine, methionine, threonine, aspartic acid, glutamic acid, glutamine and ornithine, arginine, and citrulline itself were measured. No other changes in measured parameters were observed, and study subjects tolerated 4-week-long oral supplementation of ornithine hydrochloride or citrulline without treatment-related adverse events. A clinical, no-observed-adverse-effect-level (NOAEL) of ornithine hydrochloride and citrulline supplementation in healthy adult males was determined to be 12 g/day and 24 g/day (4 weeks), respectively.

Electrostatically-sprayed carbon electrodes for high performance organic complementary circuits.

Organic thin-film transistors (OTFTs) are promising building blocks of flexible printable electronic devices. Similar to inorganic FETs, OTFTs are heterostructures consisting of metals, insulators, and semiconductors, in which nanoscale interfaces between different components should be precisely engineered. However, OTFTs use noble metals, such as gold, as electrodes, which has been a bottleneck in terms of cost reduction and low environmental loading. In this study, we demonstrate that graphite-based carbon electrodes can be deposited and patterned directly onto an organic single-crystalline thin film via electrostatic spray coating. The present OTFTs exhibited reasonably high field-effect mobilities of up to 11 cm2 V-1 s-1 for p-type and 1.4 cm2 V-1 s-1 for n-type with no significant deterioration during electrostatic spray processes. We also demonstrate two significant milestones from the viewpoint of material science: a complementary circuit, an inverter consisting of p- and n-type OTFTs, and an operatable metal-free OTFT composed of fully carbon-based materials. These results constitute a key step forward in the further development of printed metal-free integrated circuits.

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.

Prune Juice Containing Sorbitol, Pectin, and Polyphenol Ameliorates Subjective Complaints and Hard Feces While Normalizing Stool in Chronic Constipation: A Randomized Placebo-Controlled Trial.

INTRODUCTION: The aim of this study was to determine the effectiveness of prune juice on chronic constipation. METHODS: We conducted a double-blind, randomized, placebo-controlled trial in Japanese subjects with chronic constipation. RESULTS: Prune intake significantly decreased hard and lumpy stools while increasing normal stool and not increasing loose and watery stools. Prune intake also ameliorated subjective complaints of constipation and hard stools, without alteration of flatulence, diarrhea, loose stools, or urgent need for defecation. There were no adverse events or laboratory abnormalities of liver or renal function after prune intake. DISCUSSION: Prune juice exerted an effective and safe natural food therapy for chronic constipation.

Potential Biomarkers for Chronic Seasonal Heat Stress in Kagoshima Berkshire Pigs Reared in the Subtropical Region.

Introduction: Potential biomarkers for chronic seasonal heat stress in Kagoshima Berkshire pigs reared in the subtropical region were investigated by comparing the biomarker changes in the summer (a period of chronic heat stress) and winter (a thermoneutral period) seasons. Material and Methods: Pigs were allocated to summer- and winter-finishing cohorts, 12 each. The evaluations included assessment of carcass traits and internal organs' normality carried out at the time of slaughter, and measurement of biomarkers in whole blood: derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential as markers of oxidative stress, and serum amyloid A and albumin/globulin (A/G) ratio as markers of acute and chronic inflammation, respectively. Results: The summer-finished pigs reared under subtropical field conditions showed lower carcass quality than the winter-finished pigs, indicating a potential adverse effect of summer temperatures on the swine industry. Marginal changes were observed in d-ROMs and the A/G ratio between the summer- and winter-finishing cohorts. Conclusion: The results demonstrate that d-ROMs and the A/G ratio could be used as sensitive markers for heat stress under field conditions.

The presence of adjacent others facilitates interpersonal neural synchronization in the left prefrontal cortex during a simple addition task.

The hyperscanning technique, that is, simultaneous measurement of neural signals in more than one person, is a powerful research tool for understanding humans' social interactions. In recent years, many studies have investigated interpersonal neural synchronization during various types of communication processes. However, there has been little focus on the impact of the presence of others without explicit social interaction, despite the mere presence of others having been suggested as influencing behavior. In this study, we clarify whether neural signals during a self-paced, repeated, addition task are synchronized when another individual is adjacent without direct interaction. Twenty pairs of participants were measured using a hyperscanning approach with near-infrared spectroscopy. The results show that interpersonal neural synchronization of the task-related signal in the left forehead region was enhanced under the condition of being adjacent to another participant. By contrast, a significant decrease in neural synchronization in the center of the forehead region, where increased neural synchronization is often reported in explicit communication, was observed. Thus, the results indicate that the adjacency of others modulates interpersonal neural synchronization in the task-related signal, and the effect on cognitive processing is different from that of explicit social interaction.

NGS-identified miRNAs in Canine Mammary Gland Tumors Show Unexpected Expression Alterations in qPCR Analysis.

BACKGROUND/AIM: Canine mammary gland tumors (MGTs), as a potential model of human breast cancer, have a well-defined histological classification system. MicroRNA (miRNA) expression is a key part of the molecular signatures of both MGTs and human breast cancer, although the signatures alone do not yet provide a sufficient basis for definitive diagnosis. In this study, we investigated the association between miRNA expression patterns and histological classification. MATERIALS AND METHODS: Mammary gland tissue was collected from healthy dogs (n=7) and dog patients (n=80). Further samples (n=5) were obtained from established MGT cell lines. We targeted miRNAs differentially expressed in metastatic tumor tissue versus non-metastatic and normal tissue. A subset of samples was analyzed using small RNA next generation sequencing (NGS) with subsequent qPCR. RESULTS: Six differentially expressed miRNAs were selected from the NGS analysis and submitted for large-scale qPCR. The large-scale qPCR analysis revealed greater alternations in miRNA expression. Large-scale analysis, based on 79 samples, revealed a hierarchical clustering based on selected miRNAs that did not strikingly match the histopathological subtype classification. CONCLUSION: We successfully investigated the large-scale miRNA expression pattern in canine MGT and provided the whole miRNA expression. The selected miRNA demonstrated that there is no straightforward mapping between molecular signatures and histological classification of canine MGTs at the miRNA level.

A clinical case of a subepiglottic cyst in a Japanese Black calf.

A 36-day-old Japanese Black calf exhibited wheezing associated with dyspnea from birth. Arterial blood gas analysis revealed a low oxygen partial pressure of 51 mmHg, low oxygen saturation of 83%, and high carbon dioxide partial pressure of 58.8 mmHg. Computed tomography, endoscopy, and ultrasonography showed cyst formation under the epiglottis. When the cyst was aspirated under ultrasonic guidance to secure the airway, 30 ml of viscous white turbid content was aspirated. The cyst shrank immediately after aspiration, but the wheezing and respiratory symptoms resumed 7 days after aspiration. Therefore, the cyst was surgically removed from the ventral side of the neck. No cyst remodeling was observed 30 days after surgical removal.

Long non-coding RNA and transfer RNA-derived small fragments in exosomes are potential biomarkers for canine oral melanoma.

Novel small non-coding RNAs (sRNAs) represent an emerging line of research in both human and canine oncology, due to their diverse regulatory and functional roles. Novel sRNAs are regarded as distinct from microRNAs, although both are part of the exosomal cargo. Recently, we reported on exosomal miRNAs as biomarkers for canine melanoma; however, it is unknown if novel sRNAs hold similar potential. Accordingly, we aimed to identify and validate novel sRNAs as potential biomarkers of canine oral melanoma, as part of our larger project on sequencing small exosomal RNA for this disease. Next generation sequencing revealed several differentially expressed novel sRNAs in exosomes from two melanoma cell lines (KMeC and LMeC) when compared with reference exosomes (from tumour-free dogs). Among these novel sRNAs, long noncoding RNA fragments, tRNA-derived fragments, snoRNAs and snRNAs were abundantly expressed. We selected four novel sRNAs upregulated in each cell line, and validated their aberrant expression with qPCR. In analysis using plasma-derived exosomes from melanoma patients, six out of the eight selected novel sRNAs showed significantly elevated expression. Receiver operating curve (ROC) analysis showed that one long non-coding RNA-derived small fragment (ENSCAFT00000069599.1) and one transfer RNA-derived small fragment (tRNA-Ala-TGC-5-1) have more than 85% sensitivity and specificity for differentiating melanoma patients from tumour-free dogs. Therefore, we consider that novel sRNAs may serve as candidate biomarkers to facilitate more accurate diagnosis of canine oral melanoma in clinical settings.