Effects of prenatal exposure to diclofenac sodium and saline on the optic nerve of 4- and 20-week-old male rats: a stereological and histological study

We investigated the effects of diclofenac sodium (DS) on development of the optic nerve in utero. Pregnant female rats were separated into three groups: control, saline treated and DS treated. Offspring of these animals were divided into 4-week-old and 20-week-old groups. At the end of the 4th and 20th weeks of postnatal life, the animals were sacrificed, and right optic nerves were excised and sectioned for ultrastructural and stereological analyses. We demonstrated that both DS and saline produced structural and morphometric changes in the total axon number and density of axons, but decreased the myelin sheath thickness in male optic nerves. All ultrastructural and morphometric features were well developed in 20-week-old rats. We showed that development of the optic nerve continues during the early postnatal period and that some compensation for exposure to deleterious agents in utero may occur during early postnatal life.     

Biochemical,Stabilization and Crystallization Studies on a Molecular Chaperone (PaoD) Involved in the Maturation of Molybdoenzymes

Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C₄mim]Cl and [C₂OHmim]PF₆ and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference – nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.     

Heterogeneity of silica and glycan-epitope distribution in epidermal idioblast cell walls in Adiantum raddianum laminae

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.     

Synthesis of novel pyrazoline derivatives and the evaluation of death mechanisms involved in their antileukemic activity

Malignant neoplasms are one of the leading causes of death worldwide and hematologic malignancies, including acute leukemia (AL) is one of the most relevant cancer types. Current available chemotherapeutics are associated with high morbidity and mortality rates, therefore, the search for new molecules with antitumor activity, specific and selective for neoplastic cells, became a great challenge for researchers in the oncology field. As pyrazolines stand out in the literature for their great variety of biological activities, the aim of this study was to synthesize and evaluate the antileukemic activity of five new pyrazoline derivatives. All pyrazolines showed adequate physicochemical properties for a good oral bioavailability. The two unpublished and most effective pyrazoline derivatives have been selected for further experiments. These compounds are highly selective for leukemic cells when compared to non-neoplastic cells and did not cause lysis on human red blood cells. Additionally, selected pyrazolines induced cell cycle arrest at G0/G1 phase and decreased cell proliferation marker KI67. Apoptotic cell death induced by selected pyrazolines was confirmed by morphological analysis, assessment of phosphatidylserine residue exposure and DNA fragmentation. Several factors indicate that both intrinsic and extrinsic apoptosis occurred. These were: increased FasR expression; the predominance of Bax in relation to Bcl-2; the loss of mitochondrial membrane potential; AIF release; decreased expression of survivin (an antiapoptotic protein); and the activation of caspase-3. The selected pyrazolines were also found to be cytotoxic against neoplastic cells collected from the peripheral blood and bone marrow of patients with different subtypes of acute leukemia.     

Network analysis of temporal functionalities of the gut induced by perturbations in new-born piglets

Background

Evidence is accumulating that perturbation of early life microbial colonization of the gut induces long-lasting adverse health effects in individuals. Understanding the mechanisms behind these effects will facilitate modulation of intestinal health. The objective of this study was to identify biological processes involved in these long lasting effects and the (molecular) factors that regulate them. We used an antibiotic and the same antibiotic in combination with stress on piglets as an early life perturbation. Then we used host gene expression data from the gut (jejunum) tissue and community-scale analysis of gut microbiota from the same location of the gut, at three different time-points to gauge the reaction to the perturbation. We analysed the data by a new combination of existing tools. First, we analysed the data in two dimensions, treatment and time, with quadratic regression analysis. Then we applied network-based data integration approaches to find correlations between host gene expression and the resident microbial species.

Results

The use of a new combination of data analysis tools allowed us to identify significant long-lasting differences in jejunal gene expression patterns resulting from the early life perturbations. In addition, we were able to identify potential key gene regulators (hubs) for these long-lasting effects. Furthermore, data integration also showed that there are a handful of bacterial groups that were associated with temporal changes in gene expression.

Conclusion

The applied systems-biology approach allowed us to take the first steps in unravelling biological processes involved in long lasting effects in the gut due to early life perturbations. The observed data are consistent with the hypothesis that these long lasting effects are due to differences in the programming of the gut immune system as induced by the temporary early life changes in the composition and/or diversity of microbiota in the gut.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1733-8) contains supplementary material, which is available to authorized users.     

1,000 structures and more from the MCSG

Background  

The Midwest Center for Structural Genomics (MCSG) is one of the large-scale centres of the Protein Structure Initiative (PSI). During the first two phases of the PSI the MCSG has solved over a thousand protein structures. A criticism of structural genomics is that target selection strategies mean that some structures are solved without having a known function and thus are of little biomedical significance. Structures of unknown function have stimulated the development of methods for function prediction from structure.     

Evolving DNA motifs to predict GeneChip probe performance

Background  

Affymetrix High Density Oligonuclotide Arrays (HDONA) simultaneously measure expression of thousands of genes using millions of probes. We use correlations between measurements for the same gene across 6685 human tissue samples from NCBI's GEO database to indicated the quality of individual HG-U133A probes. Low correlation indicates a poor probe.