The Trophic Effect of Ouabain on Retinal Ganglion Cell is Mediated by EGF Receptor and PKC δ Activation

It was already shown that ouabain treatment can stimulate PKC isoenzymes leading to the activation of intracellular pathways involved in cell survival, growth and proliferation. We have previously demonstrated that ouabain or PMA treatment increases retinal ganglion cell survival, an effect mediated by PKC activation. The aim of this work was to investigate the role of EGF receptors in the ouabain effect and also to study which PKC isoform is activated by treatment with ouabain and PMA. Our results show that 2.5 μM tyrphostin, 1.0 μM PP1, 4.0 μM U73122, 1.0 μM JNK inhibitor V and 2.0 μM rottlerin blocked the ouabain effect indicating an involvement of receptors for EGF, Src, PLC, JNK and PKC δ respectively. The effect of PMA was only abolished when cultures were treated with rottlerin or with the JNK inhibitor suggesting the involvement of PKC δ and JNK. These results indicate that PKC δ could be a key regulator of retinal ganglion cell survival.     

DNA Barcoding of Neotropical Sand Flies (Diptera,Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.     

Host-Parasite Interactions in Chagas Disease: Genetically Unidentical Isolates of a Single Trypanosoma cruzi Strain Identified In Vitro via LSSP-PCR

The present study aims at establishing whether the diversity in pathogenesis within a genetically diverse host population infected with a single polyclonal strain of Trypanosoma cruzi is due to selection of specific subpopulations within the strain. For this purpose we infected Swiss mice, a genetically diverse population, with the polyclonal strain of Trypanosoma cruzi Berenice-78 and characterized via LSSP-PCR the kinetoplast DNA of subpopulations isolated from blood samples collected from the animals at various times after inoculation (3, 6 and 12 months after inoculation). We examined the biological behavior of the isolates in acellular medium and in vitro profiles of infectivity in Vero cell medium. We compared the characteristics of the isolates with the inoculating strain and with another strain, Berenice 62, isolated from the same patient 16 years earlier. We found that one of the isolates had intermediate behavior in comparison with Berenice-78 and Berenice-62 and a significantly different genetic profile by LSSP-PCR in comparison with the inoculating strain. We hereby demonstrate that genetically distinct Trypanosoma cruzi isolates may be obtained upon experimental murine infection with a single polyclonal Trypanosoma cruzi strain.     

Oogenesis in the Apogamous Fern Pteris cretica

The events that characterize egg formation and maturation inPteris cretica were investigated using transmission electronmicroscopy and electron microscope microprobe analysis. Theydid not differ significantly from those described for sexuallyreproducing ferns. The significance of these findings is discussedin relation to current theories concerning phase change in ferns. Pteris cretica, fern, apogamy, agamospory, transmission electron microscopy, oogenesis     

Reprogramming to a pluripotent state modifies mesenchymal stem cell resistance to oxidative stress

Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood–derived mesenchymal stem cells (mb‐iPSC), embryonic stem cells (H9) and adult menstrual blood–derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb‐iPSC were 10‐fold less resistant to H₂O₂, which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb‐iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb‐iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb‐iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb‐iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS.     

Effect of foliar‐applied potassium silicate on coffee leaf infection by Hemileia vastatrix

Coffee leaf rust, caused by Hemileia vastatrix, is the most devastating disease of coffee. Since limited information is available in the literature on silicon (Si) affecting plant diseases in coffee, this study was designed to investigate foliar application of potassium silicate (PS), a source of soluble (Si), on infection process of coffee leaf rust at the microscopic level. The foliar Si concentration for plants sprayed with water and PS has no significant difference (0.24 and 0.30 dag kg^?1, respectively). X‐ray microanalysis indicated that the deposition of Si on the leaves of the plants that were sprayed with PS was greater in comparison to the leaf samples from the plants sprayed with water. Rust severity on leaves of plants sprayed with water or sprayed with PS reached 44% and 32%, respectively, at 36 days after inoculation (dai). Plates of polymerised PS were observed on the leaf surfaces of the plants sprayed with the product, in contrast to its absence on the leaf surfaces of plants sprayed with water. At 36 dai, a greater number of uredia were observed on the leaf surfaces of plants sprayed with water in comparison to the leaf surfaces of plants sprayed with PS. On fractured leaf tissues that were sprayed with PS, less fungal colonisation was observed in comparison to the leaves of plants sprayed with water. In conclusion, the results of this study suggest that the effect of foliar‐applied Si on the control of the coffee leaf rust development may be attributed to the physical role of the polymerised PS, its osmotic effect against urediniospores germination, or both.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.