Effects of in vitro exposure of sheep ovarian tissue to zearalenone and matairesinol on preantral follicles.

The aim of this study was to evaluate the effect of 1 µmol/l zearalenone (ZEN) and 1 µmol/l matairesinol (MAT), alone or in combination, on the morphology of in vitro-cultured ovarian preantral follicles. Ovaries from four adult sheep were collected at a local slaughterhouse and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN. The plant lignan MAT alone did not maintain the morphology of the ovarian follicles; its combination with ZEN counteracted the negative effects observed when follicles were cultured in the presence of the mycotoxin alone. However, MAT was not able to promote the in vitro development of the ovarian follicles.

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

In vitro exposure of sheep ovarian tissue to the xenoestrogens zearalenone and enterolactone: Effects on preantral follicles.

The aim of this study was to evaluate the effect of 1 µmol/L zearalenone (ZEN) and 1 µmol/L enterolactone (ENL), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 10 sheep were collected at a local abattoir and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN, whereas that of cultured secondary follicles was improved by ENL. However, the combination of ENL with ZEN impaired the quality of primary and secondary follicles. Both ZEN and ENL induced apoptosis, but only ZEN was responsible for oocyte autophagy. None of these xenoestrogens affected endoplasmic reticulum stress as observed by the unaltered expression of ERP29. Differently from ZEN, ENL increased the expression of the efflux transporter ABCG2. In conclusion, although ENL can counteract the negative effects of ZEN on primordial and primary follicles, this positive effect is not similar to that observed in ovarian tissue cultures in the presence of ENL alone.

A Brazilian regional basic diet-induced chronic malnutrition drives liver inflammation with higher ApoA-I activity in C57BL6J mice.

Malnutrition is still considered endemic in many developing countries. Malnutrition-enteric infections may cause lasting deleterious effects on lipid metabolism, especially in children living in poor settings. The regional basic diet (RBD), produced to mimic the Brazilian northeastern dietary characteristics (rich in carbohydrate and low in protein) has been used in experimental malnutrition models, but few studies have explored the effect of chronic RBD on liver function, a central organ involved in cholesterol metabolism. This study aimed to investigate whether RBD leads to liver inflammatory changes and altered reverse cholesterol metabolism in C57BL6/J mice compared to the control group, receiving a standard chow diet. To evaluate liver inflammation, ionized calcium-binding adapter protein-1 (IBA-1) positive cell counting, interleukin (IL)-1ß immunohistochemistry, and tumor necrosis factor (TNF)-α and IL-10 transcription levels were analyzed. In addition, we assessed reverse cholesterol transport by measuring liver apolipoprotein (Apo)E, ApoA-I, and lecithin-cholesterol acyltransferase (LCAT) by RT-PCR. Furthermore, serum alanine aminotransferase (ALT) was measured to assess liver function. RBD markedly impaired body weight gain compared with the control group (P<0.05). Higher hepatic TNF-α (P<0.0001) and IL-10 (P=0.001) mRNA levels were found in RBD-challenged mice, although without detectable non-alcoholic fatty liver disease. Marked IBA-1 immunolabeling and increased number of positive-IBA-1 cells were found in the undernourished group. No statistical difference in serum ALT was found. There was also a significant increase in ApoA mRNA expression in the undernourished group, but not ApoE and LCAT, compared with the control. Altogether our findings suggested that chronic RBD-induced malnutrition leads to liver inflammation with increased ApoA-I activity.

Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review.

Egg yolk constitutes a relevant alternative source of antibodies. It presents some advantages over mammalian serum immunoglobulins regarding productivity, animal welfare and specificity. The main immunoglobulin present in avian blood (IgY) is transmitted to their offspring and accumulates in egg yolks, which enables the non-invasive harvesting of high amounts of antibodies. Moreover, due to structural differences and phylogenetic distance, IgY is more suitable for diagnostic purposes than mammalian antibodies, since it does not react with certain components of the human immune system and displays greater avidity for mammalian conserved proteins. IgY has been extensively used in health researches, as both therapeutic and diagnostic tool. This article aims to review its applications in both human and veterinary health.

Evaluation of the toxicity and hypoglycemic effect of the aqueous extracts of Cnidoscolus quercifolius Pohl.

Diabetes mellitus is one of the most common chronic degenerative diseases, and it is estimated to increase worldwide to around 415 million and to impact 642 million in 2040. Research shows that some plants are sources of bioactive compounds against diabetes. Thus, the objective of this work was to evaluate the oral toxicity and the hypoglycemic effect of the aqueous extract of the leaves of Cnidoscolus quercifolius Pohl. Diabetes was induced in Swiss mice with streptozotocin and the mice were treated with an aqueous extract of C. quercifolius leaves for a period of 30 days. Phytochemical analysis showed that the extract was rich in flavonoids, catechins and triterpenoid, which did not show any mortality and behavioral alterations in mice treated with 200, 1000, and 2000 mg/kg body weight of the extract for 14 days. Histopathological analysis of organs (kidney, pancreas, liver) from mice treated with the 2000 mg/kg extract revealed no architectural change. In the present study, we found a 29% reduction in glucose levels in animals receiving 200 mg/kg body weight. These results are very promising because they showed that C. quercifolius had a hypoglycemic effect and did not present oral toxicity, thus being a new source of compounds for the control of diabetes.

Hypolipidemic activity of P-methoxycinnamic diester (PCO-C) isolated from Copernicia prunífera against Triton WR-1339 and hyperlipidemic diet in mice.

Carnauba wax is extracted from the leaves of the Copernicia prunífera and contains approximately 80% of esters in its composition. The purpose of the present study was evaluate the hypolipidemic effect of p-methoxycinnamic diesters (PCO-C) extracted from Copernicia prunífera in a model of acute and chronic dyslipidemia in mice. The levels of total cholesterol and triglycerides were significantly reduced plasma levels in PCO-C at the dose of 100mg/kg in a model of acute and chronic dyslipidemia. Histological studies showed that PCO-C has no hepatotoxic effect and reduces hepatic steatosis in animals that consumed hyperlipidemic ration. Thus, it was concluded that PCO-C isolated from Copernicia Prunifera was effective in reducing total cholesterol and triglyceride levels in both dyslipidemia induction models. The finding indicates that PCO-C might be beneficial in treatment of hyperlipidemia and atherosclerosis.

A Brazilian regional basic diet-induced chronic malnutrition drives liver inflammation with higher ApoA-I activity in C57BL6J mice

Malnutrition is still considered endemic in many developing countries. Malnutrition-enteric infections may cause lasting deleterious effects on lipid metabolism, especially in children living in poor settings. The regional basic diet (RBD), produced to mimic the Brazilian northeastern dietary characteristics (rich in carbohydrate and low in protein) has been used in experimental malnutrition models, but few studies have explored the effect of chronic RBD on liver function, a central organ involved in cholesterol metabolism. This study aimed to investigate whether RBD leads to liver inflammatory changes and altered reverse cholesterol metabolism in C57BL6/J mice compared to the control group, receiving a standard chow diet. To evaluate liver inflammation, ionized calcium-binding adapter protein-1 (IBA-1) positive cell counting, interleukin (IL)-1β immunohistochemistry, and tumor necrosis factor (TNF)-α and IL-10 transcription levels were analyzed. In addition, we assessed reverse cholesterol transport by measuring liver apolipoprotein (Apo)E, ApoA-I, and lecithin-cholesterol acyltransferase (LCAT) by RT-PCR. Furthermore, serum alanine aminotransferase (ALT) was measured to assess liver function. RBD markedly impaired body weight gain compared with the control group (P<0.05). Higher hepatic TNF-α (P<0.0001) and IL-10 (P=0.001) mRNA levels were found in RBD-challenged mice, although without detectable non-alcoholic fatty liver disease. Marked IBA-1 immunolabeling and increased number of positive-IBA-1 cells were found in the undernourished group. No statistical difference in serum ALT was found. There was also a significant increase in ApoA mRNA expression in the undernourished group, but not ApoE and LCAT, compared with the control. Altogether our findings suggested that chronic RBD-induced malnutrition leads to liver inflammation with increased ApoA-I activity.

A disposable chitosan-modified carbon fiber electrode for dengue virus envelope protein detection.

A chitosan-modified carbon fiber electrode (CFE) for dengue virus envelope protein (DENV) was developed. Antibodies against DENV were covalently immobilized on the chitosan (CHIT) matrix after activation with sodium periodate. Cyclic voltammetries and scanning electron microscopies analysis were performed to monitor steps involved in the CFE surface modification. Amperometric response of the competitive immunoassays was generated by hydrogen peroxide reaction with the peroxidase conjugated to DENV and 2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as mediator. The immunosensor showed a lower limit of detection for DENV (0.94 ng mL(-1)) than previously described and a linear range from 1.0 to 175 ng mL(-1), in concentration levels clinically relevant for dengue virus diagnosis. The intra- and inter-assay were respectively 5.8% and 3.6%. The unique and simple design of this immunoassay format provides an economical alternative for the manufacture of other sensitive sensors.

Colonization of cashew plants by Lasiodiplodia theobromae: microscopical features.

Lasiodiplodia theobromae is a phytopathogenic fungus causing gummosis, a threatening disease for cashew plants in Brazil. In an attempt to investigate the ultrastructural features of the pathogen colonization and its response to immunofluorescence labeling, light, confocal and electron microscope studies were conducted on different severity scale patterns of diseased plants. Lasiodiplodia-antisera was checked for cross reactivity against common cashew plants fungi. Optical microscopy analysis revealed a longitudinally sectioned hyphae located within the xylem vessels, showing an extensive hyphal development in the secondary xylem tissue. SEM images demonstrated that the fungus was found in some asymptomatic samples, particularly within the xylem vessels as confirmed by the optical images. Symptomatic sample images showed an extensive distribution of the fungus along the secondary xylem, within the vessels, infecting xylem parenchyma. A closer look in the secondary xylem parenchyma reveals a heavy and profuse invasion of the cells with a distinguishable cell wall disintegration and fully hyphae dispersal. There was no reactivity of Lasiodiplodia-antisera against mycelial extracts of Colletotrichum gloeosporioides, Phomopsis anardii and Pestalotiopsis guepinii. Following incubation of sections with the polyclonal antisera, the hyphae were intensely and regularly labeled. Rays, vessels and parenchyma cells were the preferred pathway for L. theobromae colonization. Artificial infection provides the information that the vascular cylinder is undoubtedly employed and used by the fungus for hyphae distribution. Immunofluorescence assay employed in situ was applied and the polyclonal antisera produced was able to recognize the fungus and proved to be a sensitive technique to detect it.

Estudo preliminar toxicológico, antibacteriano e fitoquímico do extrato etanólico das folhas de Jatropha mollissima (Pohl) Baill. (pinhão-bravo, Euphorbiaceae), coletada no Município de Tauá, Ceará, Nordeste Brasileiro / Toxicological, antibacterial, and phytochemical preliminary study of the ethanolic extract of Jatropha mollissima (Pohl) Baill (pinhão-bravo, Euphorbiaceae) leaves, collected in Tauá, Ceará, Northeastern Brazil

RESUMO A cada dia, cepas bacterianas estão tornando-se resistentes a diversos antibióticos, o que faz necessária a busca de novas substâncias eficazes para o tratamento de doenças. Desta forma, este trabalho reporta o estudo preliminar toxicológico, antibacteriano e fitoquímico do extrato etanólico das folhas de Jatropha mollissima (pinhão-bravo, Euphorbiaceae), coletada no Município de Tauá, Ceará, Nordeste Brasileiro. Inicialmente, realizou-se o teste de toxicidade do extrato contra Artemia salina. Na sequencia, foi realizado o ensaio antibacteriano contra quatro cepas bacterianas Gram-negativas (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Hafnia alvei ATCC 51873, Klebsiella pneumoniae ATCC 13883) e uma cepa Gram-positiva (Enterococcus faecalis ATCC 29212). Finalmente, fez-se a análise fitoquímica preliminar do extrato ativo para detecção das principais classes de metabólitos especiais. Como resultado, o extrato etanólico das folhas de J. mollissima se mostrou tóxico para Artemia salina, pois apresentou CL50 igual a 406,02 μg/mL. Quanto à ação antibacteriana, o extrato se mostrou ativo contra a bactéria Gram-positiva Enterococcus faecalis ATCC 29212, apresentando moderada atividade antibacteriana (halo de inibição igual a 7,03 mm). Evidenciou-se no extrato bioativo a presença de cumarinas, fenóis, taninos, flavonoides (flavonóis e flavanonas), alcaloides e esteroides, ambas as classes reportadas como antimicrobianos. Portanto, esse extrato tem potencial para ser usado na produção de fármacos contra infecções causadas por bactérias Gram-positivas. No entanto, as informações direcionam estudos futuros para o isolamento e identificação dos compostos bioativos, monitorados sob a ação antibacteriana mais expressiva.

ABSTRACT Each day, bacterial strains are becoming more resistant to various antibiotics, which requires the search for new effective substances for the treatment of diseases. Thus, this study reports the toxicological, antibacterial, and phytochemical preliminary study of the ethanolic extracts of Jatropha mollissima (pinhão-bravo, Euphorbiaceae) leaves, collected in Tauá, Ceará, Northeast of Brazil. Initially, we performed the toxicity testing of the extract against Artemia salina. Then, we conducted the antibacterial assay against four Gram-negative bacterial strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Hafnia alvei ATCC 51873, Klebsiella pneumoniae ATCC 13883), and one Gram-positive strain (Enterococcus faecalis ATCC 29212). Finally, we carried out the preliminary phytochemical analysis of the active extract to detect the main classes of special metabolites. As a result, the ethanolic extract of J. mollissima leaves was toxic to Artemia salina, because it presented LC50 equal to 406.02 µg/mL. Regarding antibacterial action, the extract was active against the Gram-positive bacteria Enterococcus faecalis ATCC 29212, with moderate antibacterial activity (inhibition zone equal to 7.03 mm). The bioactive extract had the presence of coumarins, phenols, tannins, flavonoids (flavanols and flavonones), alkaloids and steroids, both classes reported as antimicrobials. Therefore, this extract has the potential to be used in the production of drugs against infections caused by Gram-positive bacteria. However, these information require further studies for the isolation and identification of bioactive compounds, monitored under the more expressive antibacterial action.

Antiviral activities of extracts and phenolic components of two Spondias species against dengue virus

In recent years, the search for natural plant products to fight viral diseases has been increasing. In this work, two Spondias species, namely S. mombin and S. tuberosa, found in Ceará state (Brazil), and their main phenolic components were evaluated against dengue virus. In vitro antiviral tests were performed against type-2 dengue virus by the MTT method and standard cytopathic effect reduction assay in C6/36 cells. Cytotoxicity was also evaluated by MTT. The presence of phenolic compounds quercetin, rutin, and ellagic acid in plant extracts was characterized by HPLC analysis. Both Spondias species extracts and components were nontoxic to the cells whereas rutin and quercetin displayed relevant antiviral activity with IC50 of 362.68 µg/mL and 500 µg/mL, respectively.

Perfil hematológico e avaliação eletroforética das proteínas séricas de cães com cinomose / Hematological profile and electrophoretic evaluation of serum proteins of dogs with canine distemper

The hematological and serum proteins electrophoretic profiles of 13 dogs with distemper (Lentz inclusion body in leukocytes) were studied. The most frequent hematological findings were: normocitic normocromic anemia (61%), leukopenia (46%), left shount (54%), trombocytopenia (69%) and lymphopenia (85%). Electrophoretic analysis of serum proteins showed hypoproteinemia (54%), with reduced albumin and increased alfa-2 globulin. These findings can be used to support the clinical diagnosis of canine distemper.