Recurrent gene mutations detected in canine mast cell tumours by next generation sequencing.

Genetic causes of canine mast cell tumours (MCTs), except for mutations in the KIT gene detected in some MCTs, are generally unknown. We used whole exome sequencing to reveal mutation spectra in canine MCTs. We detected somatic mutations in 87 genes including 10 genes recognized as human cancer drivers. Besides KIT, 14 other genes were recurrently mutated. Subsequently, we performed next generation sequencing of a panel of 50 selected genes in additional MCT samples. In this group, the most frequently altered gene was GNB1 showing a recurrent dinucleotide substitution at position of Gly116 in 30% of the MCT samples (n = 6/20) and Ile80 substitution accompanied by a splice region mutation in one case. We extended the study by analysis of the above mentioned GNB1 regions in additional MCT samples by Sanger sequencing, and assessed the overall prevalence of GNB1 mutations to 17.3% (n = 14/81), which is similar to the prevalence of KIT alterations. Our results indicate that GNB1 mutations are probably involved in canine MCT pathogenesis in both cutaneous and subcutaneous MCT cases. As opposed to KIT alterations, the presence of GNB1 mutations did not negatively affect survival times, and our data even showed a trend towards positive prognosis. If our results are confirmed in a larger number of MCTs, an extension of molecular testing of canine MCTs by GNB1 analysis would help to refine the molecular stratification of MCTs, and become useful for targeted treatment strategies.

Mutation and methylation status of KIT and TP53 in canine cutaneous and subcutaneous mast cell tumours.

Cutaneous and subcutaneous mast cell tumours (MCTs) are counted among the most frequent cancers in dogs. However, the genetic aetiology of their development is still mostly unknown, with the exception of KIT and tumor protein p53 (TP53 ) mutations reported in less than a half of cutaneous MCTs. In subcutaneous MCTs, no gene alterations were previously detected. We analysed KIT and TP53 mutations in cutaneous and subcutaneous MCTs, and identified methylated CpG sites in KIT and TP53 promoters and adjacent exon 1 regions. The mutation analysis focused on KIT exons 8, 9 and 11, and TP53 exons 5-8, and revealed mutations in 26% and 7% cutaneous MCT cases, respectively. Moreover, we report a first case of KIT mutation ever detected in subcutaneous MCTs. KIT exon 11 mutations and high Kiupel and Patnaik grades were associated with reduced survival in this study. Both KIT and TP53 gene were generally unmethylated in canine cutaneous MCTs. A sporadic methylation of the CpG positions in KIT promoter and adjacent exon 1 was detected in 70.4% of cutaneous and 82% of subcutaneous MCTs. A sporadic methylation of the CpG positions in the TP53 promoter and exon 1 was observed in 36.8% of the analysed cutaneous MCT samples. Only in two subcutaneous MCTs, we observed more than 30% of clones showing KIT methylation at the CpG positions 13 or 14. The CpG position 14 is involved in a predicted binding site for Sp1 transcription factor. However, the significance of KIT promoter methylation at this specific position needs further evaluation.

Prevalence and prognostic value of c-kit and TP53 mutations in canine mast cell tumours.

Cutaneous mast cell tumours (MCT) are among the most frequent malignancies in dogs. Their clinical behaviour is highly variable and, with the exception of mutations in the c-kit gene, little is known about their genetic aetiology. The mutational status of the c-kit exons 8, 9 and 11, and exons 5-8 of the TP53 gene was analysed to find markers for molecular stratification of MCTs and predictors of clinical outcome. Mutations in the c-kit gene were detected in 19.5% (n = 8/41) samples and their presence was significantly associated with the high histopathological grade (P = 0.038). Mutations in the DNA binding domain of the TP53 gene were found in 14.6% (n = 6/41) of the analysed MCTs, and their frequency was similar in low and high grade MCTs (P > 0.05). TP53 mutations were not useful prognostic factors in this sample of canine cutaneous MCTs.

Structural and copy number chromosome abnormalities in canine cutaneous mast cell tumours.

Mast cell tumours (MCTs) are the most common skin tumours in dogs. Their clinical behaviour is variable and their aetiology remains largely unknown. We performed a metaphase fluorescence in situ hybridisation (FISH) with whole chromosome painting probes, and interphase FISH with BAC probes for 14 cancer-related genes to reveal clonal structural chromosome rearrangements and copy number variants (CNVs) in canine cutaneous MCTs. The metaphase FISH performed in three MCTs revealed several clonal monosomies and trisomies and two different chromosome rearrangements. No centric fusions were detected. The interphase FISH showed a variety of low frequency CNVs for the individual cancer-related genes. The heterogeneous character of the detected abnormalities indicates increased chromosome instability in canine MCTs. The clonal gain of chromosome 11 was detected in 81% (13/16) of the MCTs. Further research is needed to evaluate the significance of this abnormality as prognostic factor for the survival time or recurrence risk assessments in canine cutaneous MCTs.

Inhalation of ZnO Nanoparticles: Splice Junction Expression and Alternative Splicing in Mice.

Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 × 104 and 1.93 × 106 NP/cm3 for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFNγ, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 × 106 NP/cm3. In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.

Multibiomarker Responses of Juvenile Stages of Zebrafish (Danio rerio) to Subchronic Exposure to Polycyclic Musk Tonalide.

Synthetic polycyclic musks, widely used as additives in personal care products, are present in both biotic and abiotic matrices of the aquatic environment at concentrations of ng/l to µg/l. Although they are determined at comparatively low concentrations, these levels are biologically relevant and pose a significant growing risk as stressors to aquatic organisms. The purpose of our study was to evaluate the effects of 28-day-long exposure to polycyclic musk tonalide in zebrafish juvenile stages (Danio rerio) using selected biomarkers. Environmentally relevant concentrations of tonalide caused significant changes in selected enzyme activities in the experimental groups exposed to the highest concentrations. The activity of glutathione S-transferase and lipid peroxidation increased significantly (p < 0.05) after exposure to the highest concentration (50,000 ng/l) compared with the control. A similar trend was observed in catalase activity; there was a significant increase (p < 0.05) after exposure to two highest concentrations of tonalide (5000 and 50,000 ng/l). In addition, a statistically significant decrease (p < 0.05) in glutathione reductase activity was found in the lowest test concentration of tonalide (50 ng/l). None of the tested concentrations resulted in histopathological changes in liver, kidney, skin, or gill. Furthermore, no effects on body weight, body length, specific growth rate, and behavior were observed. Our results showed that tonalide exposure induced profound changes in the activities of antioxidant and detoxifying enzymes, such changes representing an adaptive response of the fish organism to tonalide toxicity.

Prenylated flavonoid morusin protects against TNBS-induced colitis in rats.

Morusin is a prenylated flavonoid isolated from the root bark of Morus alba. Many studies have shown the ability of flavonoids to act as anti-inflammatory agents. The aim of this study was to evaluate the effect of morusin on experimentally colitis induced by 2,4,6-trinitrobenzensulfonic acid in Wistar rats and to compare it with sulfasalazine, a drug conventionally used in the treatment of inflammatory bowel disease. Morusin was administered by gavage at doses of 12.5, 25, or 50 mg/kg/day for five days. The colonic tissue was evaluated macroscopically, histologically, and by performing immunodetection and zymographic analysis to determine the levels of antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)], interleukin (IL)-1ß, and transforming growth factor (TGF)-ß1 and the activities of matrix metalloproteinases (MMP) 2 and 9. The tissue damage scores were significantly reduced with increasing dose of morusin, however efficacy was not demonstrated at the highest dose. At the dose of 12.5 mg/kg, morusin exerted therapeutic effectivity similar to that of sulfasalazine (50 mg/kg). This was associated with significant reduction of TGF-ß1 levels and MMP2 and MMP9 activities, and slight reduction of IL-1ß. Our results suggest that morusin possesses therapeutic potential for the treatment of chronic inflammatory diseases.

Inhaled Cadmium Oxide Nanoparticles: Their in Vivo Fate and Effect on Target Organs.

The increasing amount of heavy metals used in manufacturing equivalently increases hazards of environmental pollution by industrial products such as cadmium oxide (CdO) nanoparticles. Here, we aimed to unravel the CdO nanoparticle destiny upon their entry into lungs by inhalations, with the main focus on the ultrastructural changes that the nanoparticles may cause to tissues of the primary and secondary target organs. We indeed found the CdO nanoparticles to be transported from the lungs into secondary target organs by blood. In lungs, inhaled CdO nanoparticles caused significant alterations in parenchyma tissue including hyperemia, enlarged pulmonary septa, congested capillaries, alveolar emphysema and small areas of atelectasis. Nanoparticles were observed in the cytoplasm of cells lining bronchioles, in the alveolar spaces as well as inside the membranous pneumocytes and in phagosomes of lung macrophages. Nanoparticles even penetrated through the membrane into some organelles including mitochondria and they also accumulated in the cytoplasmic vesicles. In livers, inhalation caused periportal inflammation and local hepatic necrosis. Only minor changes such as diffusely thickened filtration membrane with intramembranous electron dense deposits were observed in kidney. Taken together, inhaled CdO nanoparticles not only accumulated in lungs but they were also transported to other organs causing serious damage at tissue as well as cellular level.

Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats.

Diplacone (1) and mimulone (2), two geranylated flavanones, have previously shown anti-inflammatory and antiradical activity in vitro. The present study aimed to evaluate their activity in vivo on a model of colitis induced in Wistar rats by an oral administration of dextran sulfate sodium (DSS). Diplacone (1) and mimulone (2) were administered at a bolus dose of 25mg/kg by gastric gavage 48 and 24h prior to the induction of colitis by DSS and every 24h on the following days of the experiment. The effect of the treatment was assessed by monitoring the disease activity index (DAI), histopathological examination, evaluation of the weight and length of the colon and by analysis of the levels and activities of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP2), superoxide dismutase-2 (SOD2), and catalase (CAT) in the inflamed tissue. Administration of the test compounds prior and after induction of colitis ameliorated the symptoms of colitis (diarrhea, presence of the blood in the stool) and delayed their onset. The ability of compounds 1 and 2 to reduce the levels of COX-2 and to increase the ratio of pro-MMP2/MMP2 activity correlates with the values of the DAI. The lowering of the levels of the antioxidant enzymes SOD2 and CAT reflects the ability of the test compounds to scavenge reactive oxygen species.

The effect of mycotoxin deoxynivalenol on haematological and biochemical indicators and histopathological changes in rainbow trout (Oncorhynchus mykiss).

Deoxynivalenol (DON), produced by the Fusarium genus, is a major contaminant of cereal grains used in the production of fish feed. The effect of mycotoxin deoxynivalenol on rainbow trout (Oncorhynchus mykiss) was studied using a commercial feed with the addition of DON in a dose of 2 mg/kg feed. The fish (n=40) were exposed to the mycotoxin for 23 days. The trout were divided into two groups, control and experimental groups. Control groups were fed a commercial feed naturally contaminated with a low concentration of DON (225 µg/kg feed); experimental groups were fed a commercial feed with the addition of DON (1964 µg/kg feed). Plasma biochemical and haematological indices, biometric parameters, and histopathological changes were assessed at the end of the experiment. The experimental groups showed significantly lower values in MCH (P<0.05). In biochemical indices, after 23-day exposure, a significant decrease in glucose, cholesterol (P<0.05), and ammonia (P<0.01) was recorded in the experimental group compared to the control group. Our assessment showed no significant changes in biometric parameters. The histopathological examination revealed disorders in the caudal kidney of the exposed fish. The obtained data show the sensitivity of rainbow trout (O. mykiss) to deoxynivalenol.

The effects of mycotoxin deoxynivalenol (DON) on haematological and biochemical parameters and selected parameters of oxidative stress in piglets.

OBJECTIVES: Deoxynivalenol (DON) - trichothecene mycotoxin, is frequently detected in high concentrations in cereals in the temperate region of Europe. The aim of this study was to determine the effect of DON in feed on haematological and biochemical parameters and on oxidative stress in piglets. METHODS: Two concentrations of DON in feedstuff for pigs were chosen: 0.6 mg/kg (group C) and 2.0 mg/kg (group M). Twelve weaned pigs were used in each group. Pigs were fed with naturally contaminated feed for 4 weeks. On days 14, 21 and at the end of the experiment (day 28) samples of blood were taken to determine haematological parameters, plasma biochemical parameters, ceruloplasmin activity and FRAP (ferric reducing ability of plasma). RESULTS: The haematological variables did not show changes in response to contaminated diet with exception of the mean corpuscular volume, which was significantly decreased at the end of the experiment in the group M. A significant increase of alkaline phosphatase activity (140%, p<0.01) was found in the group M compared to the group C at the end of the experiment. A significant decrease was found on the day 21 in FRAP (85%, p<0.001) and on the day 28 in ceruloplasmin (75%, p<0.01) in the group M compared to the group C. CONCLUSIONS: The decrease of FRAP and ceruloplasmin indicate a lowered ability of organism to scavenge reactive oxygen species. The higher concentration of DON in feedstuffs had a negative influence on the antioxidant ability of piglet's plasma.

Outbreak of Mycobacterium avium subsp. avium infection in one flock of domestic pigeons.

An outbreak of Mycobacterium avium subsp. avium infection was diagnosed in one breed of domestic pigeons (Columba livia f. domestica) in the Czech Republic. Nodular granulomatous lesions were found in 42 (9.7%) pigeons of the 435 examined; histopathologic examination of livers with gross lesions of mycobacteriosis from 15 randomly selected pigeons revealed granulomatous inflammation typical for avian mycobacteriosis in all samples. Direct Ziehl-Neelsen (ZN) microscopy and conventional culture were performed for a total of 117 liver samples (42 pigeons with nodular lesions, 55 randomly selected pigeons without nodular lesions, and 20 randomly selected squabs). Acid-fast bacilli were observed in 19 (16.2%), and conventional culture yielded growth of M. a. avium in 40 (34.2%) liver samples. A triplex quantitative real-time PCR assay based on the IS901 detection system was performed successfully in 115 liver samples and revealed M. a. avium in 63 (54.8%) of them. Mycobacterium a. avium was also detected in two squabs. Eight domestic rabbits (Oryctolagus cuniculus f. domestica) living in the breeding facility were also examined. Pyogranulomatous lesions were only found in one adult male rabbit. At necropsy, both direct ZN microscopy and culture gave negative results for mycobacteria in all examined rabbit tissues. Mycobacterium a. avium was diagnosed in a liver sample of one juvenile rabbit using triplex qPCR, suggesting that M. a. avium infection can occur as early as juvenile animals.

Mycobacterium avium Subsp. avium and Salmonella enterica serotype Typhimurium var. Copenhagen phage type DT2 in pigeons.

We report on a coinfection of Mycobacterium avium subsp. avium and Salmonella enterica serotype Typhimurium var. Copenhagen phage type DT2 in pigeons from one flock, from which squabs were occasionally consumed by humans. Triplex quantitative real-time PCR and culture methods were used for M. a. avium detection in livers and culture method was used for the detection of Salmonella sp. in samples of liver and caecum of 33 examined birds. M. a. avium was detected in a total of 31 (93.9%) and Salmonella Typhimurium in a total of 11 (33.3%) pigeons. Coinfection with both pathogens was found in 10 (30.3%), infection with Salmonella Typhimurium alone in 1 (3.0%), and infection with M. a. avium alone in 21 (63.7%) pigeons. Neither pathogen was detected in one pigeon. There was no difference in clinical symptoms exhibited by pigeons infected by M. a. avium and/or Salmonella Typhimurium. All Salmonella Typhimurium isolates were sensitive to all 15 antimicrobials tested. According to these results we emphasize good heat treatment of consumed squabs.

Endogenous development of Hemolivia mauritanica (Apicomplexa: Adeleina: Haemogregarinidae) in the marginated tortoise Testudo marginata (Reptilia: Testudinidae): evidence from experimental infection.

Six young tortoises Testudo marginata Schoepff, 1792 were experimentally infected with Hemolivia mauritanica (Sergent et Sergent, 1904). The prepatent period ranged from 6 to 8 weeks. Young, smaller, club-like forms (6-9 x 3-6 Am) of gametocytes appeared in the peripheral blood first, whereas mature, elongated, cylindrical forms (9-12 x 5-7 Am) were detected after 1-2 weeks and predominated during later patency. Three of the infected tortoises were euthanized and dissected to study the endogenous stages. Meronts occurred in the cells of the reticulo-endothelial system and in the erythrocytes; these were observed mostly in parenchymatous organs. Mature forms measured 14.2 x 9.3 microm and contained 7-12 merozoites. Cysts with two (exceptionally one) cystozoites were also found predominantly in parenchymatous organs and measured 14.8 x 7.9 microm. Pathological changes attributable to Hemolivia were mild and limited to liver and kidneys. The role of individual developmental stages of haemogregarines is discussed with respect to evolution of heteroxenous life cycle and long-term persistence of parasites in their intermediate hosts.