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In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P < 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines.

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The use of tyrosine kinase inhibitors (TKI) has been growing in veterinary oncology and in the past few years several TKI have been tested in dogs. However, different from human medicine, we lack strategies to select patients to be treated with each TKI. Therefore, this study aimed to screen different tumor subtypes regarding TKI target immunoexpression as a predictor strategy to personalize the canine cancer treatment. It included 18 prostatic carcinomas, 36 soft tissue sarcomas, 20 mammary gland tumors, 6 urothelial bladder carcinomas, and 7 tumors from the endocrine system. A total of 87 patients with paraffin blocks were used to perform immunohistochemistry (IHC) of human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptors 1 (EGFR1), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet derived growth factor receptor beta (PDGFR-ß), c-KIT, and extracellular signal-regulated kinase 1/2 (ERK1/ERK2). The immunohistochemical screening revealed a heterogeneous protein expression among histological types with mesenchymal tumors showing the lowest expression level and carcinomas the highest expression. We have demonstrated by IHC screening that HER2, EGFR1, VEGFR-2, PDGFR-ß and ERK1/ERK2 are commonly overexpressed in dogs with different carcinomas, and KIT expression is considered relatively low in the analyzed samples.

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The present study focused on comparing the gene expression profiles of different mouse models of prostate cancer, focusing on the TRAMP transgenic model and its derived cell lines and extending the comparisons to relevant genetically engineered mouse models and human prostate cancer datasets. Employing RNA sequencing, we examined different levels of prostate cancer aggressiveness from the original TRAMP cells to the TRAMP-C2 (TC2) derived cell line and extending to the aggressive TC2-Ras (TC2R) cells and tumors. TC2R acquire the ability to grow in bone tissue upon implantation, unlike the parental TC2 cells. Analysis identified upregulated genes in cell cycle regulation, immune response, and mitotic processes in TRAMP compared to wild-type tissues. TC2 cells exhibited unique gene profiles enriched in ECM organization and tissue development pathways, while TC2R cells showed increased cytokine signaling and motility genes, with decreased ECM and immune response pathways. In vivo TC2R models demonstrated enhanced ECM organization and receptor tyrosine kinase signaling in tumors, notably enriching immune processes and collagen degradation pathways in intratibial tumors. Comparative analysis among mouse and human datasets showed overlaps, particularly in pathways relating to mitotic cycle regulation, ECM organization, and immune interactions. A gene signature identified in TC2R tumors correlated with aggressive tumor behavior and poor survival in human datasets. Further immune cell landscape analysis of TC2R tumors revealed altered T cell subsets and macrophages, confirmed in single-cell RNA-seq from human samples. TC2R models thus hold significant promise in helping advance preclinical therapeutics, potentially contributing to improved prostate cancer patient outcomes.

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Splenic tumors are very common in dogs, and canine hemangiosarcoma (HSA) is one of the most important malignant splenic tumors. Surgery followed by chemotherapy (anthracycline-based protocols) is recommended for treating canine HSA; however, patients still do not achieve long-term survival. Therefore, this research aimed to assess vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor-ß (PDGFR-ß) gene expression in formalin-fixed tissues, evaluate the quality of mRNA for quantitative polymerase chain reaction (qPCR) analysis and identify drug repositioning candidates based on VEGFR-2 and PDGFR-ß. qPCR analysis identified the relative expression of heterogeneous VEGFR-2 and PDGFR-ß, with samples showing no transcripts or very low expression and those with higher relative quantification for both genes. We then used immunohistochemistry to correlate the relative quantification of VEGFR-2 and PDGFR-ß transcripts with respective higher protein expression to validate our results. In the next step, we evaluated drug repositioning candidates and identified small molecule inhibitors (i.e. sorafenib) and natural compounds (curcumin and resveratrol) with the ability to block VEGFR-2 and PDGFR-ß genes. Overall, our results indicated that VEGFR-2 and PDGFR-ß expression is highly variable among canine HSA samples and different drugs can block the expression of both genes. Therefore, a personalized approach could be useful for selecting anti-VEGFR-2 and PDGFR-ß therapies and both genes are potential candidates for future oncological panels.

Os tumores esplênicos são muito comuns em cães, e o hemangiosarcoma (HSA) é um dos tumores esplênicos malignos mais importantes em cães. A cirurgia seguida de quimioterapia (protocolos baseados em antraciclinas) é a abordagem terapêutica mais recomendada para o tratamento da HSA canino; no entanto, os pacientes ainda não alcançam longa sobrevida após tratamento. Portanto, esta pesquisa teve como objetivo avaliar a expressão do receptor do fator de crescimento endotelial vascular-2 (VEGFR-2) e do receptor do fator de crescimento derivado de plaquetas-ß (PDGFR-ß) em tecidos fixados em formalina e identificar candidatos ao reposicionamento de medicamentos baseado na expressão desses genes. A análise qPCR identificou a expressão relativa heterogênea de VEGFR-2 e PDGFR-ß, com amostras sem transcritos ou com expressão muito baixa ou amostras com alta quantificação relativa para ambos os genes. Em seguida, foi realizada o exame imuno-histoquímico para correlacionar a quantificação relativa dos transcritos de VEGFR-2 e PDGFR-ß com a respectiva maior expressão proteica para validar nossos resultados. Na próxima etapa, avaliamos candidatos ao reposicionamento de medicamentos e identificamos inibidores de moléculas pequenas (ou seja, sorafenibe) e compostos naturais (curcumina e resveratrol) com capacidade de bloquear os genes VEGFR-2 e PDGFR-ß. No geral, nossos resultados indicaram que a expressão de VEGFR-2 e PDGFR-ß é altamente variável entre amostras caninas de HSA e diferentes drogas podem bloquear a expressão de ambos os genes. Portanto, uma abordagem personalizada poderia ser útil para selecionar terapias anti-VEGFR-2 e PDGFR-ß e ambos os genes são potenciais candidatos para futuros painéis oncológicos.

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This study aimed to evaluate the effect of applying oxidized cassava starch-based edible coatings with addition of lemongrass essential oil emulsion on 'Palmer' mangoes stored under refrigeration. A completely randomized design was used, arranged in a 5 × 3 factorial scheme, with five types of coatings and three evaluation times. The evaluated postharvest quality parameters consisted of weight loss, pulp and peel firmness, biochemical transformations related to pigments, and pulp and peel coloration of mango. The application of edible coatings with a 0.9 % EO concentration resulted in delayed fruit ripening, evidenced mainly by a 7.25 % reduction in weight loss, a 29.23 % increase in soluble solids content, and a 24.15 % decrease in total chlorophyll, when compared to uncoated fruits, which showed 19.8 %, 48.66 %, and 82.00 %, respectively, over the storage period. This effect was also evident in the angle Hue (°h) measurement, with uncoated fruits showing a decrease of 32.2 %. The antimicrobial effect and absence of anthracnose symptoms were observed in the fruits in which the coating with 0.9 % EO was applied. Therefore, biodegradable coating with the addition of 0.9 % emulsion EO, can be used as postharvest treatments for maintenance quality of 'Palmer' mangoes during refrigerated storage.

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American cutaneous leishmaniasis is a disease caused by protozoa of the genus Leishmania. Currently, meglumine antimoniate is the first-choice treatment for the disease. The limited efficacy and high toxicity of the drug results in the necessity to search for new active principles. Nanotechnology is gaining importance in the field, since it can provide better efficacy and lower toxicity of the drugs. The present study aimed to synthesize, characterize, and evaluate the in vitro leishmanicidal and antileukemic activity of bismuth nanoparticles (BiNPs). Promastigotes and amastigotes of L. (V.) guyanensis and L. (L.) amazonensis were exposed to BiNPs. The efficacy of the nanoparticles was determined by measurement of the parasite viability and the percentage of infected cells, while the cytotoxicity was characterized by the colorimetry. BiNPs did not induce cytotoxicity in murine peritoneal macrophages and showed better efficacy in inhibiting promastigotes (IC50 < 0.46 nM) and amastigotes of L. (L.) amazonensis. This is the first report on the leishmanicidal activity of Bi-based materials against L. (V.) guayanensis. BiNPs demonstrated significant cytotoxic activity against K562 and HL60 cells at all evaluated concentrations. While the nanoparticles also showed some cytotoxicity towards non-cancerous Vero cells, the effect was much lower compared to that on cancer cells. Treatment with BiNPs also had a significant effect on inhibiting and reducing colony formation in HL60 cells. These results indicate that bismuth nanoparticles have the potential for an inhibitory effect on the clonal expansion of cancer cells.

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Cutaneous squamous cell carcinoma (cSCC) is a neoplasm type often diagnosed in dogs. However, studies focused on further investigating its molecular biology, mainly biomarkers to help implementing new therapies, remain scare in the literature. Thus, immunostaining and the gene expression of epidermal growth factor receptors (HER1 and HER2) in canine cSCC presenting different cell differentiation degrees were herein assessed. Thirty-two (32) canine cSCC were selected, classified based on to their cell differentiation degree and subjected to immunohistochemical study to assess HER1 and HER2 immunostaining intensity and distribution. In addition, HER1 and HER2 gene expression was investigated through real-time PCR. Membranous and cytoplasmic immunostaining were observed in both markers. HER2 prevailed in poorly differentiated cSCC; there was positive protein expression correlation between both markers. Mean HER1 gene expression was higher in moderately differentiated, whereas mean HER2 gene expression was higher in poorly differentiated cSCC. Moreover, there was gene expression correlation between markers, regardless of cell differentiation degree. Thus, HER2 protein immunostaining and gene expression were higher in poorly differentiated canine cSCC and it enabled understanding that increase observed in this epidermal growth factor receptor is proportional to this neoplasm's cell differentiation degree in canine species. Results in the current study helped better understanding canine cSCC's molecular biology; however, it is relevant studying other markers aiming to investigate signaling pathways.

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Nanocapsules provide selective delivery and increase the bioavailability of bioactive compounds. In this study, we examined the anticancer and immunomodulatory potential of Fridericia chica (crajiru) extract encapsulated in nanocapsules targeting myeloid leukemias. Nanocapsules containing crajiru (nanocapsules-CRJ) were prepared via interfacial polymer deposition and solvent displacement. Size and polydispersity were measured by dynamic light scattering. Biological assays were performed on leukemia cell lines HL60 and K562 and on non-cancerous Vero cells and human PBMC. The anticancer activity was evaluated using cytotoxicity and clonogenic assays, while the immunomodulatory activity was evaluated by measuring the levels of pro- and anti-inflammatory cytokines in PBMC supernatants treated with concentrations of nanocapsules-CRJ. Nanocapsules-CRJ exhibited significant cytotoxic activity against HL60 and K562 cells at concentrations ranging from 0.75 to 50 µg/mL, with the greatest reductions in cell viability observed at 50 µg/mL (p < 0.001 for HL60; p < 0.01 for K562), while not affecting non-cancerous Vero cells and human PBMCs. At concentrations of 25 µg/mL and 50 µg/mL, nanocapsules-CRJ reduced the formation of HL60 and K562 colonies by more than 90% (p < 0.0001). Additionally, at a concentration of 12 µg/mL, nanocapsules-CRJ induced the production of the cytokines IL-6 (p = 0.0002), IL-10 (p = 0.0005), IL-12 (p = 0.001), and TNF-α (p = 0.005), indicating their immunomodulatory potential. These findings suggest that nanocapsules-CRJ hold promise as a potential therapeutic agent with both cytotoxic and immunomodulatory properties.

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The aberrant activation of HER2 has a pivotal role in bone metastasis implantation and progression in several tumor types, including prostate cancer (PC). Trastuzumab and other anti-HER2 therapies, such as lapatinib, have been used in human breast cancer HER2 positive. Although HER2 overexpression has been reported in PC, anti-HER2 therapy response has revealed conflicting results. We investigated the potential of lapatinib in inhibiting cell migration and inducing apoptosis in two human (LNCaP and PC3) and two canine PC cell lines (PC1 and PC2). Cell migration and apoptosis were evaluated by Annexin V/PI analysis after lapatinib treatment. The transcriptome analysis of all cell lines before and after treatment with lapatinib was also performed. We found increased apoptosis and migration inhibition in LNCaP cells (androgen-sensitive cell line), while PC1, PC2, and PC3 cells showed no alterations after the treatment. The transcriptome analysis of LNCaP and PC3 cell lines showed 158 dysregulated transcripts in common, while PC1 and PC2 cell lines presented 82. At the doses of lapatinib used, we observed transcriptional modifications in all cell lines. PI3K/AKT/mTOR pathway were enriched in human PC cells, while canine PC cells showed enrichment of tyrosine kinase antitumor response and HER2-related pathways. In canine PC cells, the apoptosis failed after lapatinib treatment, possibly due to the downregulation of MAPK genes. Prostate cancer cells insensitive to androgens may be resistant to lapatinib through PI3K gene dysregulation. The association of lapatinib with PI3K inhibitors may provide a more effective antitumor response and clinical benefits to PC patients.

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Background: Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent ß-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. Methods: In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Results: Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Conclusion: Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research.

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The Epstein-Barr virus (EBV) is a ubiquitous human pathogen linked to various diseases, including infectious mononucleosis and multiple types of cancer. To control and eliminate EBV, the host's immune system deploys its most potent defenses, including pattern recognition receptors, Natural Killer cells, CD8+ and CD4+ T cells, among others. The interaction between EBV and the human immune system is complex and multifaceted. EBV employs a variety of strategies to evade detection and elimination by both the innate and adaptive immune systems. This demonstrates EBV's mastery of navigating the complexities of the immunological landscape. Further investigation into these complex mechanisms is imperative to advance the development of enhanced therapeutic approaches with heightened efficacy. This review provides a comprehensive overview of various mechanisms known to date, employed by the EBV to elude the immune response, while establishing enduring latent infections or instigate its lytic replication.

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The giant anteater (Myrmecophaga tridactyla) is a vulnerable species in South America and is considered endangered or near extinction in Central America. Therefore, studies describing the reproductive characteristics of this species are pivotal for its conservation. Thus, this study aimed to provide a morphological description of the female reproductive tissues of this species. We collected tissue samples from six female giant anteaters and performed gross, morphological, and histochemical analyses. Five adult subjects and one juvenile were included in the study. In the ovary, classifications were made according to the follicle and oocyte sizes: primordial, primary, secondary, early antral, or antral. Typical follicles with a single oocyte surrounded by a simple or stratified layer of cubic epithelium, atretic follicles, corpora lutea, corpora albicans, and ovarian cysts were also observed. No ovarian lesions were observed. By contrast, endometritis, metritis, mucometra, and endometrial cysts were identified in the uterus. Uterine alterations in these subjects were frequent and could affect reproduction.

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Background: Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. Methods: In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Results: Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Conclusion: Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research.

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The tumor microenvironment is considered one of the main players in cancer development and progression and may influence the behavior of cancer cells. Periostin (POSTN) is an extracellular matrix protein, and its main functions are induction of fibrillogenesis, fibroblastic cell proliferation and migration, enhancing regeneration in normal tissue, and promoting metastasis in case of neoplasia. POSTN has already been studied in humans in several normal tissues, inflammatory processes, and neoplasms, revealing an important role in tumor progression in various types of cancer, such as colon, lung, head and neck, breast, ovarian, and prostate. In these latter, high levels of POSTN are usually associated with a more aggressive tumor behavior, tumor advanced stages, and poor prognosis, while in human bladder urothelial carcinoma (BUC), unlike in most tumors, POSTN expression seems to be downregulated. The expression of this marker has been poorly investigated in veterinary medicine; thus, this study aimed to immunohistochemically investigate the presence and the intensity of POSTN expression in canine BUCs and to determine a possible relationship between POSTN expression and histopathological features such as mitotic count and muscular and vascular invasions. For the present retrospective study, archived samples from 45 canine BUCs and 6 non-neoplastic canine bladders were considered for histological evaluation and immunohistochemical examination for the expression of POSTN. POSTN expression was semi-quantitatively assessed considering both the percentage of the neoplastic stroma positive for POSTN and the intensity of the immunohistochemical labeling. Histologically, 38 out of 45 tumors were papillary and 7 out of 45 were non-papillary. All tumors were infiltrating, being that 21 were muscle-invasive, and a significant correlation between this feature and vascular invasion emerged (P = 0.0001). In normal bladder tissue, as reported in humans, a thick and strongly positive belt of POSTN was visible, and in canine BUCs, stating that the expression is comparable with human benign as well as malignant bladder tissue, a general decrease in POSTN expression was observed except for a strongly labeled ring of POSTN observed around some neoplastic nodules infiltrating the muscle layer. Moreover, POSTN expression and mitotic count were significatively inversely correlated (P = 0.0015). The fact that POSTN protein is less expressed in urothelial carcinomas than in the normal bladder supports what was reported in human BUCs and, together with the negative correlation between mitotic count and protein expression that emerged in the present retrospective study, encourages further prospective follow-up studies to verify the possible role of POSTN in canine BUCs as a prognostic marker, and also as a possible target for the development of future anticancer therapies.

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Osteopontin (OPN) is a protein synthesized by a large number of cells, and its overexpression has been associated with the development and prognosis of cancer. OPN overexpression has been claimed to be a marker for the development of bone metastasis in human cancers, but no prior research has investigated the association between OPN expression and the metastasis of canine mammary gland tumors (MGTs) and prostate cancer (PC). Therefore, we investigated OPN expression in MGTs and PC samples from 50 canine patients with or without metastasis (bone vs. other sites). Higher OPN expression was detected in primary tumor samples from animals with bone metastasis than in those without bone involvement (p = 0.0321). In MGT samples, a significantly lower survival rate was observed in patients with higher OPN expression (p = 0.0171). In animals with PC, there was a strong trend toward lower survival in animals with positive OPN expression; however, this trend was not statistically significant (p = 0.0779). From these findings, it can be concluded that OPN may be a promising target for future MGTs and PC studies because of its role in enhancing cell invasion and metastasis.

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The discovery and development of effective novel compounds is paramount in oncology for improving cancer therapy. In this study, we developed a new derivative of spiroindolone (7',8'-Dimethoxy-1',3'-dimethyl-1,2,3',4'-tetrahydrospiro[indole-3,5'- pyrazolo[3,4-c]isoquinolin]-2-one) and evaluated its anticancer- and immunomodulatory potential in a vitro model of chronic leukemia. We utilized the chronic leukemia cell line K562, as well as non-cancerous peripheral blood mononuclear cells (PBMC) and Vero cells (kidney epithelium of Cercopithecus aethiops). We assessed the cytotoxicity of the compound using the MTT assay, and performed cell cycle assays to determine its impact on different stages of the cell cycle. To evaluate its antineoplastic activity, we conducted a colony formation test to measure the effect of the compound on the clonal growth of cancer cells. Furthermore, we evaluated the immunomodulatory activity of the compound by measuring the levels of pro and anti-inflammatory cytokines. The study findings demonstrate that the spiroindolone-derived compound exerted noteworthy cytotoxic effects against K562 cells, with an IC50 value of 25.27 µg/mL. Additionally, it was observed that the compound inhibited the clonal proliferation of K562 cells while displaying minimal toxicity to normal cells. The compound exhibited its antiproliferative activity by inducing G2/M cell cycle arrest, preventing the entry of K562 cells into mitosis. Notably, the compound demonstrated an immunomodulatory effect by upregulating the production of cytokines IL-6 and IL-12/23p40. In conclusion, the spiroindolone-derived compound evaluated in this study has demonstrated significant potential as a therapeutic agent for the treatment of chronic myeloid leukemia. Further investigations are warranted to explore its clinical applications.

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Aural plaques have been linked to Equus caballus papillomavirus (EcPV). Ten types of EcPVs have already been described; however, only EcPVs 1, 3, 4, 5, and 6 have been observed in association with aural plaques. Accordingly, the objective of this study was to evaluate the presence of EcPVs in equine aural plaque samples. A total of 29 aural plaque samples (from 15 horses) were collected and assessed for the presence of the DNA of these EcPVs by PCR. Additionally, 108 aural plaque samples used in previous research were evaluated for the presence of EcPVs 8 and 9. Previously described primers were used for PCR to detect EcPVs 1 to 8, and specific primers were designed for EcPV 9. Minigenes were synthesized and used as a positive control in the PCRs for the undetected EcPVs. EcPVs 2, 7, 8, and 9 were not detected in any of the evaluated samples, suggesting that these viral types are not involved in the etiology of the equine aural plaque in Brazil. EcPV 6 was the most prevalent (81%), followed by EcPVs 3 (72%), 4 (63%) and 5 (47%), which reinforces the idea that these viruses play an important role in the etiology of the equine aural plaque in Brazil.

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