Análise molecular e ultraestrutural de espermatozóides caprinos oriundos de animais infectados naturalmente e experimentalmente com o CAEV.

O presente trabalho teve como objetivo realizar uma análise molecular e ultraestrutural em espermatozóides caprinos, afim de se averiguar a presença ou não do CAEV em sêmen de animais infectados naturalmente e experimentalmente. Para isto, foi coletado sêmen de 12 machos caprinos, sendo 4 machos infectados naturalmente com o CAEV, 4 experimentalmente e 4 negativos pelos testes de imunodifusão em gel de agarose (IDGA) e Reação em cadeia da polimerase (PCR), sendo este último grupo utilizado somente como controle. A coleta de sêmen foi realizada utilizando-se o método da vagina artificial. Foram realizadas seis coletas de cada animal e após cada coleta as amostras seminais foram destinadas às técnicas de PCR e Microscopia Eletrônica de Transmissão. Das 23 amostras seminais analisadas oriundas de animais infectados naturalmente apenas uma amostra apresentou positividade para o teste de PCR. Das 22 amostras provenientes de animais infectados artificialmente não se observou nenhuma amostra positiva, não tendo se observado nenhuma diferença estatística entre os grupos. Com relação às amostras analisadas ao microscópio eletrônico de transmissão, apenas em uma das amostras oriunda de animal infectado naturalmente, foi observado presença de vacúolos na porção da peça intermediária, porém, não foi observada nenhuma partícula viral no seu interior. O que demonstrou que espermatozóides de caprinos infectados naturalmente com o CAEV apresenta indícios de susceptibilidade para este vírus

Antimicrobial effect of farnesol, a Candida albicans quorum sensing molecule, on Paracoccidioides brasiliensis growth and morphogenesis

<p>Abstract</p> <p>Background</p> <p>Farnesol is a sesquiterpene alcohol produced by many organisms, and also found in several essential oils. Its role as a quorum sensing molecule and as a virulence factor of <it>Candida albicans </it>has been well described. Studies revealed that farnesol affect the growth of a number of bacteria and fungi, pointing to a potential role as an antimicrobial agent.</p> <p>Methods</p> <p>Growth assays of <it>Paracoccidioides brasiliensis </it>cells incubated in the presence of different concentrations of farnesol were performed by measuring the optical density of the cultures. The viability of fungal cells was determined by MTT assay and by counting the colony forming units, after each farnesol treatment. The effects of farnesol on <it>P. brasiliensis </it>dimorphism were also evaluated by optical microscopy. The ultrastructural morphology of farnesol-treated <it>P. brasiliensis </it>yeast cells was evaluated by transmission and scanning electron microscopy.</p> <p>Results</p> <p>In this study, the effects of farnesol on <it>Paracoccidioides brasiliensis </it>growth and dimorphism were described. Concentrations of this isoprenoid ranging from 25 to 300 μM strongly inhibited <it>P. brasiliensis </it>growth. We have estimated that the MIC of farnesol for <it>P. brasiliensis </it>is 25 μM, while the MLC is around 30 μM. When employing levels which don't compromise cell viability (5 to 15 μM), it was shown that farnesol also affected the morphogenesis of this fungus. We observed about 60% of inhibition in hyphal development following <it>P. brasiliensis </it>yeast cells treatment with 15 μM of farnesol for 48 h. At these farnesol concentrations we also observed a significant hyphal shortening. Electron microscopy experiments showed that, despite of a remaining intact cell wall, <it>P. brasiliensis </it>cells treated with farnesol concentrations above 25 μM exhibited a fully cytoplasmic degeneration.</p> <p>Conclusion</p> <p>Our data indicate that farnesol acts as a potent antimicrobial agent against <it>P. brasiliensis</it>. The fungicide activity of farnesol against this pathogen is probably associated to cytoplasmic degeneration. In concentrations that do not affect fungal viability, farnesol retards the germ-tube formation of <it>P. brasiliensis</it>, suggesting that the morphogenesis of this fungal is controlled by environmental conditions.</p

The genome sequence of Pseudoplusia includes single nucleopolyhedrovirus and an analysis of p26 gene evolution in the baculoviruses.

Background: Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated. Results: The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX ? Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins. Conclusions: PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide

Production of bovine embryos and calves cloned by nuclear transfer using mesenchymal stem cells from amniotic fluid and adipose tissue.

201

Free Rhodium (II) citrate and rhodium (II) citrate magnetic carriers as potential strategies for breast cancer therapy

<p>Abstract</p> <p>Background</p> <p>Rhodium (II) citrate (Rh₂(H₂cit)₄) has significant antitumor, cytotoxic, and cytostatic activity on Ehrlich ascite tumor. Although toxic to normal cells, its lower toxicity when compared to carboxylate analogues of rhodium (II) indicates Rh₂(H₂cit)₄as a promising agent for chemotherapy. Nevertheless, few studies have been performed to explore this potential. Superparamagnetic particles of iron oxide (SPIOs) represent an attractive platform as carriers in drug delivery systems (DDS) because they can present greater specificity to tumor cells than normal cells. Thus, the association between Rh₂(H₂cit)₄and SPIOs can represent a strategy to enhance the former's therapeutic action. In this work, we report the cytotoxicity of free rhodium (II) citrate (Rh₂(H₂cit)₄) and rhodium (II) citrate-loaded maghemite nanoparticles or magnetoliposomes, used as drug delivery systems, on both normal and carcinoma breast cell cultures.</p> <p>Results</p> <p>Treatment with free Rh₂(H₂cit)₄induced cytotoxicity that was dependent on dose, time, and cell line. The IC₅₀values showed that this effect was more intense on breast normal cells (MCF-10A) than on breast carcinoma cells (MCF-7 and 4T1). However, the treatment with 50 μM Rh₂(H₂cit)₄-loaded maghemite nanoparticles (Magh-Rh₂(H₂cit)₄) and Rh₂(H₂cit)₄-loaded magnetoliposomes (Lip-Magh-Rh₂(H₂cit)₄) induced a higher cytotoxicity on MCF-7 and 4T1 than on MCF-10A (p < 0.05). These treatments enhanced cytotoxicity up to 4.6 times. These cytotoxic effects, induced by free Rh₂(H₂cit)₄, were evidenced by morphological alterations such as nuclear fragmentation, membrane blebbing and phosphatidylserine exposure, reduction of actin filaments, mitochondrial condensation and an increase in number of vacuoles, suggesting that Rh₂(H₂cit)₄induces cell death by apoptosis.</p> <p>Conclusions</p> <p>The treatment with rhodium (II) citrate-loaded maghemite nanoparticles and magnetoliposomes induced more specific cytotoxicity on breast carcinoma cells than on breast normal cells, which is the opposite of the results observed with free Rh₂(H₂cit)₄treatment. Thus, magnetic nanoparticles represent an attractive platform as carriers in Rh₂(H₂cit)₄delivery systems, since they can act preferentially in tumor cells. Therefore, these nanopaticulate systems may be explored as a potential tool for chemotherapy drug development.</p

Avaliação imunohistoquímica e ultraestrutural de gametas e embriões caprinos infectados com o CAEV.

O objetivo do presente estudo foi determinar a susceptibilidade dos folículos ovarianos, espermatozoides e embriões caprinos ao Vírus da Artrite Encefalite Caprina (CAEV). Para isto, foram analisados espermatozoides e folículos ovarianos pelas técnicas de imunohistoquímica e microscopia eletrônica de transmissão, antes e após protocolos de infecção in vitro com o CAEV. Foram submetidos à análise ultraestrutural, embriões caprinos produzidos in vivo, oriundos de cabras negativas e positivas para o CAEV. Nas amostras seminais, provenientes de animais tanto com infecção natural quanto dos artificialmente infectados, foi observada imunomarcação positiva dos espermatozoides, assim como alterações degenerativas na sua análise ultraestrutural. Já nas amostras de tecido ovariano, a imunomarcação foi mais discreta e identificada na região do estroma. No tocante à análise ultraestrutural, folículos e embriões se apresentaram íntegros. De acordo com esses resultados, pode-se concluir que os espermatozoides caprinos apresentaram-se infectados, assinalando a susceptibilidade dessas células ao vírus, bem como a potencialidade do CAEV ser carreado ao cerne do oócito, originando embriões infectados