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Considering the lack of consensus related to the impact of selective IL-6 receptor inhibition on bone remodeling and the scarcity of reports, especially on large bone defects, this study proposed to evaluate the biological impact of the selective inhibitor of interleukin-6 receptor (tocilizumab) in an experimental model of critical calvarial defect in rats. In this preclinical and in vivo study, 24 male Wistar rats were randomly divided into two groups (n=12/group): defect treated with collagen sponge (CG) and defect treated with collagen sponge associated with 2 mg/kg tocilizumab (TCZ). The defect in the parietal bone was created using an 8-mm diameter trephine drill. After 90 days, the animals were euthanized, and tissue samples (skull caps) were evaluated through micro-CT, histological, immunohistochemistry, cytokines, and RT-qPCR analyses. Tocilizumab reduced mononuclear inflammatory infiltration (P<0.05) and tumor necrosis factor (TNF)-α levels (P<0.01) and down-regulated tissue gene expression of BMP-2 (P<0.001), RUNX-2 (P<0.05), and interleukin (IL)-6 (P<0.05). Moreover, it promoted a stronger immunostaining of cathepsin and RANKL (P<0.05). Micro-CT and histological analyses revealed no impact on general bone formation (P>0.05). The bone cells (osteoblasts, osteoclasts, and osteocytes) in the defect area were similar in both groups (P>0.05). Tocilizumab reduced inflammatory cytokines, decreased osteogenic protein, and increased proteases in a critical bone defect in rats. Ninety days after the local application of tocilizumab in the cranial defect, we did not find a significant formation of bone tissue compared with a collagen sponge.

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Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and ß-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 µg/mL and for ß-lapachone was 0.65, 1.24, and 0.71 µg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for ß-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

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Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and β-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 μg/mL and for β-lapachone was 0.65, 1.24, and 0.71 μg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for β-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

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The objective of this study is to verify in vitro susceptibility of Pythium insidiosum against the agricultural fungicides mefenoxam and pyraclostrobin and evaluate the toxicity of both compounds. Twenty-one P. insidiosum isolates were tested against mefenoxam and pyraclostrobin using the broth microdilution method. Minimum inhibitory and oomicidal concentrations for both compounds were established. Additionally, scanning electron microscopy was performed on P. insidiosum hyphae treated with the sublethal concentration of each fungicide. The toxicity of the compounds was evaluated in vivo Caenorhabditis elegans model. The concentration to inhibit 100% of P. insidiosum growth ranged from 0·625 to 10 µg ml-1 for mefenoxam and from 0·019 to 5 µg ml-1 for pyraclostrobin. The SEM analysis revealed changes on the surface of the hyphae treated with the fungicides, suggesting possible damage caused by these compounds. There was no evidence of toxicity in vivo models. Mefenoxam and pyraclostrobin did not show toxicity at the doses evaluated and have inhibitory effects on the pathogenic oomycete P. insidiosum. However, further evaluations of their pharmacokinetics and toxicity in different animal species and possible pharmacological interactions are necessary to infer a possible use in the clinical management of pythiosis.

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Canine malignant mammary neoplasms (CMMN) exhibit behavioral variability with the patient survival time depending on several prognostic factors. In the present study, 134 CMMN were selected and different immunophenotypes and their associations with clinical and pathological parameters were identified. The tumors were classified as follows: 46% of luminal B HER2-, 34% of luminal A, 13% of triple-negative, and 7% of luminal B HER2+. Shorter specific survival time were associated with larger tumor sizes (>3.0 cm, HR=1.94; P=0.0209), lymph node metastasis or distant metastasis (HR= 2.82; P <.0001), more aggressive histological types (HR= 7.15, P<0.0001), higher histological grades (HR= 12.97 P=0.011), angiolymphatic invasion (HR=4.68, P<0.0001) and luminal B HER2 - (HR= 3.27, P<0.0001) and luminal B HER2 + (HR= 7.14 P<0.0001) immunophenotypes. In patients with lymph nodal metastasis, shorter survival times were associated with luminal immunophenotype B HER2 + (P=0.003). However, in patients without metastasis, an increased risk of death was associated with the aggressive histological type. In conclusion, the classification in our study allowed us to identify subtypes with different prognoses in canine malignant mammary tumors. Factors such as clinical stage, histological type, luminal B HER2+ subtype, and angiolymphatic invasion were the most important prognostic factors.

Neoplasias mamárias malignas caninas (CMMN) apresentam variável comportamento biológico e o tempo de sobrevida depende de diversos fatores prognósticos. Neste estudo, foram selecionadas 134 CMMN, bem como identificados diferentes imunofenótipos e suas associações com parâmetros clínicos e patológicos. Os tumores foram classificados em: 46% luminal B HER2-, 34% luminal A, 13% triplo negativo e 7% luminal B HER2+. Menores taxas de sobrevida específica foram associadas a tamanhos de tumor maiores (> 3,0cm; HR = 1,94; P = 0,0209), metástases em nodais ou a distância (HR = 2,82; P <0,0001), tipos histológicos mais agressivos (HR = 7,15; P <0,0001), graus histológicos mais elevados (HR = 12,97; P = 0,011), invasão angiolinfática (HR = 4,68; P <0,0001) e aos imunofenótipos luminal B HER2- (HR = 3,27; P <0,0001) e luminal B HER2+ ( HR = 7,14; P <0,0001). Em pacientes com estágio avançado, menor sobrevida específica foi associada ao imunofenótipo luminal B HER2+ (P = 0,003). Entretanto, em estágio inicial, um risco aumentado de óbito foi associado a tipos histológicos agressivos. Em conclusão, a classificação utilizada no presente estudo permitiu identificar subtipos com diferentes prognósticos em CMMN. Estágio clínico, tipo histológico, subtipo luminal B HER2+ e invasão angiolinfática foram os fatores prognósticos mais importantes.

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INTRODUCTION: There is a need for the development of new biomarkers for diagnosis and prognosis of ovarian cancer, which can ideally serve as targets for new therapeutic modalities and individualization of treatment. The objectives of this study were to determine the prognostic significance of the neutrophil/lymphocyte ratio in the peripheral blood of patients with ovarian cancer and tumor staging, and to associate this marker with the immune expression of a panel of cytokines. METHODS: The study included 24 patients with malignant ovarian neoplasia treated at the Pelvic Mass Outpatient Clinic of the Clinical Hospital of the Federal University of Triângulo Mineiro. The neutrophil/lymphocyte ratio was calculated as the absolute number of neutrophils divided by the absolute number of lymphocytes. Expression of the cytokines was evaluated by the immunohistochemistry method (IL2, IL5, IL6, IL8, IL10 and TNF-R1). Fisher's statistical test was used for the comparisons of immunohistochemical expression with the neutrophil/lymphocyte ratio, and the unpaired T-Test was used in the analysis of the association of this ratio with tumor staging. RESULTS: A neutrophil/lymphocyte ratio > 2.6 was significantly higher in the more advanced stages (II-IV) of malignant ovarian neoplasia (p = 0.0098). In addition, this ratio > 2.6 was associated with IL2 stromal immunostaining (1-3) (p = 0.0472). CONCLUSION: Stromal IL-2 is associated with higher a neutrophil/lymphocyte ratio, suggesting a worse prognosis in ovarian cancer and its role in tumor immunology; a neutrophil/lymphocyte ratio > 2.6 is associated with more advanced stages of malignant ovarian neoplasia.

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In this essay, we quantify the concentration of collagen fibers in broiler chickens exposed to increasing concentrations of cupuacu seed by-product. Collection of material was carried out in five chickens per treatment at 70 days old in the groups: control, 5% and 10% inclusion of cupuacu seed by-product. Fragments of Thoracic Pectoralis (PT) and Iliotibial lateralis (ITL) muscles were prepared for light and electronic microscopy. The amount of collagen fibers in the muscle groups was 1.08±0.61% in the PTC group; 6.24±2.58% in PT5% and 7.30±2.75% in PT10%. In the Iliotibial Lateralis groups, the results were 6.96±3.14% in the ITLC; 7.43±4.22% in the ITL5% and 8.66±2.35% in ITL10%. The amount of collagen fibers in the ITL5% and ITL10% groups showed no significant statistical difference. However, when compared to the ILTC group, there was a significant statistical difference. The PT muscle responds to standard nutritional changes, unlike the ILT muscle, which requires a high-nutrient formulation. The use of 5% cupuacu seed by-product has proven to be a viable alternative source of animal feed, as it promotes an increase in the concentrations of collagen fibers in the musculature of broiler chickens and is possibly the determining factor in meat texture.(AU)

Neste estudo, foram quantificadas as concentrações de fibras colágenas de frangos expostos a crescentes concentrações de farinha de cupuaçu. A coleta de material foi realizada em cinco animais por tratamento, aos 70 dias de idade, nos grupos: controle, inclusão de 5% e de 10% de farinha de cupuaçu. Fragmentos dos músculos peitoral torácico (PT) e iliotibial lateral (ITL) foram preparados para microscopia de luz e eletrônica. A quantidade de fibras colágenas nos grupos foi: 1,08±0,61% no grupo PTC; 6,24±2,58% em PT5% e 7,30±2,75% em PT10%. Nos grupos iliotibial lateral, os resultados foram: 6,96±3,14% no ITLC; 7,43±4,22% no ITL5% e 8,66±2,35% em ITL10%. A quantidade de fibras colágenas nos grupos ITL5% e ITL10% não apresentou diferença estatística significativa. No entanto, quando comparados ao grupo ILTC, houve diferença estatística significativa. O músculo PT responde a mudanças nutricionais padrão, ao contrário do músculo ILT, que requer alta formulação nutricional. O uso de 5% de farinha de cupuaçu provou ser uma fonte alternativa viável de alimentação animal, pois promove um aumento nas concentrações de fibras de colágeno na musculatura de frangos de corte e é possivelmente um fator determinante na textura da carne.(AU)

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In this essay, we quantify the concentration of collagen fibers in broiler chickens exposed to increasing concentrations of cupuacu seed by-product. Collection of material was carried out in five chickens per treatment at 70 days old in the groups: control, 5% and 10% inclusion of cupuacu seed by-product. Fragments of Thoracic Pectoralis (PT) and Iliotibial lateralis (ITL) muscles were prepared for light and electronic microscopy. The amount of collagen fibers in the muscle groups was 1.08±0.61% in the PTC group; 6.24±2.58% in PT5% and 7.30±2.75% in PT10%. In the Iliotibial Lateralis groups, the results were 6.96±3.14% in the ITLC; 7.43±4.22% in the ITL5% and 8.66±2.35% in ITL10%. The amount of collagen fibers in the ITL5% and ITL10% groups showed no significant statistical difference. However, when compared to the ILTC group, there was a significant statistical difference. The PT muscle responds to standard nutritional changes, unlike the ILT muscle, which requires a high-nutrient formulation. The use of 5% cupuacu seed by-product has proven to be a viable alternative source of animal feed, as it promotes an increase in the concentrations of collagen fibers in the musculature of broiler chickens and is possibly the determining factor in meat texture.(AU)

Neste estudo, foram quantificadas as concentrações de fibras colágenas de frangos expostos a crescentes concentrações de farinha de cupuaçu. A coleta de material foi realizada em cinco animais por tratamento, aos 70 dias de idade, nos grupos: controle, inclusão de 5% e de 10% de farinha de cupuaçu. Fragmentos dos músculos peitoral torácico (PT) e iliotibial lateral (ITL) foram preparados para microscopia de luz e eletrônica. A quantidade de fibras colágenas nos grupos foi: 1,08±0,61% no grupo PTC; 6,24±2,58% em PT5% e 7,30±2,75% em PT10%. Nos grupos iliotibial lateral, os resultados foram: 6,96±3,14% no ITLC; 7,43±4,22% no ITL5% e 8,66±2,35% em ITL10%. A quantidade de fibras colágenas nos grupos ITL5% e ITL10% não apresentou diferença estatística significativa. No entanto, quando comparados ao grupo ILTC, houve diferença estatística significativa. O músculo PT responde a mudanças nutricionais padrão, ao contrário do músculo ILT, que requer alta formulação nutricional. O uso de 5% de farinha de cupuaçu provou ser uma fonte alternativa viável de alimentação animal, pois promove um aumento nas concentrações de fibras de colágeno na musculatura de frangos de corte e é possivelmente um fator determinante na textura da carne.(AU)

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Este estudo objetiva reportar a ocorrência de apoptose in vivo induzida pelo veneno da serpente Bothrops alternatus em células musculares esqueléticas. Cinco coelhos machos, adultos, receberam 150µg/kg de veneno no músculo vasto lateral, enquanto outros cinco animais receberam 0,1% de BSA diluído em PBS no mesmo local. Após 12 horas, os animais foram eutanasiados, e amostras do local de inoculação foram coletadas para análise histopatológica. Foram evidenciadas necrose e hemorragia nas células musculares. Além disso, a análise imuno-histoquímica para identificação de caspase-3 ativada revelou marcações granulares e agregadas no citoplasma das células musculares, compatíveis com o processo de apoptose. Este é o primeiro relato que confirma o veneno de B. alternatus como causador de apoptose in vivo em células musculares esqueléticas.(AU)

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Guarana (Paullinia cupana) is habitually ingested by people in the Amazon region and is a key ingredient in various energy drinks consumed worldwide. Extension in longevity and low prevalence of chronic age-related diseases have been associated to habitual intake of guarana. Anti-aging potential of guarana was also demonstrated in Caenorhabditis elegans; however, the mechanisms involved in its effects are not clear. Herein, we investigated the putative pathways that regulate the effects of guarana ethanolic extract (GEE) on lifespan using C. elegans. The major known longevity pathways were analyzed through mutant worms and RT-qPCR assay (DAF-2, DAF-16, SKN-1, SIR-2.1, HSF-1). The possible involvement of purinergic signaling was also investigated. This study demonstrated that GEE acts through antioxidant activity, DAF-16, HSF-1, and SKN-1 pathways, and human adenosine receptor ortholog (ADOR-1) to extend lifespan. GEE also downregulated skn-1, daf-16, sir-2.1 and hsp-16.2 in 9-day-old C. elegans, which might reflect less need to activate these protective genes due to direct antioxidant effects. Our results contribute to the comprehension of guarana effects in vivo, which might be helpful to prevent or treat aging-associated disorders, and also suggest purinergic signaling as a plausible therapeutic target for longevity studies.

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