Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background

In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro.

Methods

Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 μM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples ≥ 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors.

Results

Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction ≥ 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed.

Conclusion

Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.     

Hydroxyurea-induced onychomadesis in a dog with chronic myeloid leukemia: A case report

A 12-year-old Rottweiler dog was presented with a history of prostration, weight loss and hyporexia for six months. Based on complete blood tests (hematological and biochemical analyses), bone marrow examination and imaging analysis, a diagnosis of chronic myeloid leukemia was made. Treatment with hydroxyurea at a dosage of 18?mg/kg twice daily was not effective in controlling the high count of white blood cells. Furthermore, after 35 days of hydroxyurea treatment, the animal developed onycholysis, with sloughing of the claws of the left pelvic and left thoracic limbs and exposure of the distal phalanx. Interruption of the medication was implemented, with clinical healing of the ungual lesions observed three months after initiation of the drug. White blood cells returned to normal after using cyclophosphamide. Currently, the animal is in complete remission, having a disease-free interval of 575 days without chemotherapy. To the authors' knowledge, this is the first report of hydroxyurea-induced onycholysis within a short-term period in a dog diagnosed with chronic myeloid leukemia.     

The effect of plant spatial pattern within a patch on foraging selectivity of grazing sheep

Plant spatial pattern has been considered as one of the most important factors influencing forage selection of herbivores in natural grasslands. Previous work has emphasized the effects of spatial distribution patterns of food resource at the scale of whole plant communities. Our objective was to explore whether changes in spatial patterns of food within a patchy site affected forage selection of sheep within and among patches. We conducted a manipulative experiment using three native plant species of different palatability and abundance to artificially create three different quality patches in each treatment. We compared the effects of aggregated and randomly dispersed patterns, within high, medium, and low quality patches respectively, on sheep forage selection. Effects of plant spatial patterns within a patch on sheep forage selection of the patch itself strongly depended on the patch quality. For high quality patches, random dispersion of food resources significantly decreased sheep consumption of the palatable plant within the patch. This effect was reversed in low quality patches, and was not significant in medium quality patches. Changes in plant spatial patterns within high quality patches greatly influenced sheep forage selection of other patches. However, changes in plant spatial patterns within medium or low quality patches significantly influenced foraging responses of sheep only for high quality patches. We therefore conclude that high quality resource sites are the most influential and susceptible foraging areas. Our results highlight the importance of high quality resource sites when considering grazing grassland conservation and management.     

Improved digestibility of plant ingredient‐based diets for European seabass (Dicentrarchus labrax) with exogenous enzyme supplementation

This study evaluated the effect of supplementation of a plant ingredients‐based diet with two levels of commercially digestive enzyme products: Synergen? and Natugrain^® on the apparent digestibility coefficients (ADCs) of nutrients and on digestive enzyme activities in European seabass (Dicentrarchus labrax) juveniles. A control diet was formulated with 450 g/kg CP (200 g/kg from fish meal + 800 g/kg from plant protein) and 180 g/kg CL (fish oil), and four other diets were made similar to the control but supplemented with 0.2 g/kg or 0.4 g/kg of Synergen? and Natugrain^®. Digestive enzyme activities were not affected by dietary treatments. Nevertheless, chyme pH along the intestine of fish fed the Natugrain^®‐supplemented diets was reduced. The ADC of nutrients increased with exogenous digestive enzyme supplementation, with the highest ADC values being observed for the 0.4 g/kg Natugrain^® diet. The ADC of dry matter, protein, lipids, energy and phosphorus in this diet were 26%, 6%, 4%, 16% and 63% higher, respectively, than in the control diet. Overall, diet supplementation with exogenous enzyme complexes seems to have high potential for improving the utilization of plant feedstuff‐rich diets for seabass juveniles. This study revealed that Natugrain^® was more effective than Synergen? in improving the ADC of nutrients and energy in European seabass juveniles.     

Interaction of biological nitrogen fixation with sowing nitrogen fertilization on common bean in the two seasons of cultivation in Brazil

Sowing nitrogen (N) fertilization can limit biological nitrogen fixation (BNF) reducing common bean yield. The aim of this study was to evaluate the effect of sowing N fertilization plus inoculation on the growth and yield of plants in the two seasons of cultivation in Brazil. In the dry season, N fertilization and inoculation promoted a greater shoot dry weight and higher pod number and yield than only inoculated. In contrast, in the rainy season, this treatment promoted no increase in shoot dry weight and yield compared with the inoculated alone. The number of nodules was greater for the inoculated alone treatment, but nodule weight was not affected by N fertilization in either season. Therefore, sowing N fertilization and inoculation can be an agronomic practice to achieve a higher common bean yield in the dry season, while in the rainy season, the inoculation without N fertilization can support a high yield at a lower cost.     

Histology and Immunohistochemistry of the Cardiac Ventricular Structure in the Green Turtle (Chelonia mydas)

This study describes the implications of cardiac ventricular microscopy in Chelonia mydas relating to its ability to dive. For this work, 11 specimens of the marine turtle species C. mydas found dead on the coast of Rio Grande do Norte (Northeast Brazil) were used. After necropsy, fragments of the cardiac ventricular wall were fixed in 10% buffered formaldehyde solution for 24 h and then subjected to routine processing for light and scanning electron microscopy (SEM). The ventricle in this species is formed by the epicardium, myocardium and endocardium. The subepicardial layer consists of highly vascularised connective tissue that emits septa to reinforce the myocardium surface. There is an abundant and diffuse subepicardial nerve plexus shown by immunostaining technique. The thickness of the spongy myocardium and the nature of its trabeculae varied between the heart chambers. The endocardium shows no characteristic elements of the heart conduction system. The valves have a hyaline cartilage skeleton, coated by dense irregular connective tissues characterised by elastic fibres. These findings in the green turtle ventricular microscopy are related to hypoxia resistance during diving.     

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining

Background

The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49–96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-β], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed.

Results

Eleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-β] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-β] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies.

Conclusion

The identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.     

Experience counts: Integrating spearfishers’ skills and knowledge in the evaluation of biological and ecological impacts

Fishing experience and skills are not commonly considered in recreational fishery studies. To analyse potential different biological/ecological impacts of three experience levels of spearfishers (novice, intermediate and experienced), access point surveys were conducted over a period of 10 months in São Miguel Island (Azores archipelago). Groups differed in terms of catch rate and composition, species size and vulnerability (i.e. intrinsic vulnerability index of fishes to fishing). Experienced spearfishers explored different areas along the island coast, fished deeper and farther off shore, were more selective regarding fish size and target species, reached higher catch weights and had catches with a higher mean index of vulnerability. Results suggest that catch composition and rate not only depend on fish community and ecosystem health, but also on the expertise of the fishers who operate in a given area. Consequently, scientific studies should consider fishers’ experience in the survey design and data analysis to not over‐ or underestimate their potential impact.     

The use of diatom Skeletonema costatum on aquaculture‐produced purple sea urchin (Paracentrotus lividus) larvae and post‐larvae diet

The quality of the microalgae provided on Paracentrotus lividus larvae rearing is a primordial factor having a direct (nutritional properties) and indirect (water quality) impact on growth, competence and survival. Skeletonema costatum is a diatom commonly used in the bivalve cultivation. However, the use of this diatom in P. lividus larval cultivations is poorly known. The Rhodomonas spp. is a microalgae commonly used in sea urchin larvae culture. Three different diets were tested on P. lividus larvae and post‐larvae cultivation (D1—Rhodomonas marina, D2—S. costatum, D3—mixture of both algae). Larvae fed with the D2 diet (55.8%) and D3 (39.9%) had a survival at 15 DAH higher than D1 (5.5%). The low survival in D1 could be due to the higher microbiological load on microalgae (Vibrio alginolyticus and V. pectenicide). Larvae fed with S. costatum (D2) showed a lower development than other diets. The competency index was lower for larvae fed with the D2. These results show that microalgae diversified diets contribute to a better development of P. lividus larvae. During the settlement and post‐settlement phase, there was also a lower growth of the sea urchin fed with the D2 and a higher survival for D3.