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.     

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.     

Genomics and proteomics approaches to the study of cancer-stroma interactions

Background

The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.

Methods

The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.

Results

We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR.

Conclusions

A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

     

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.     

Cooperativity in the binding of the cationic biocide polyhexamethylene biguanide to nucleic acids

The interaction between the broad-spectrum antimicrobial agent, polyhexamethylene biguanide (PHMB), and various nucleic acids was investigated. Titration of either single- or double-stranded 100-bp DNA, or mixed-molecular weight marker DNA, or tRNA with PHMB caused precipitation of a complex between nucleic acid and PHMB in which the nucleotide/biguanide ratio was always close to unity. Binding of PHMB was highly cooperative, with apparent Hill coefficients 10.3-14.6. When a fluorescent derivative of PHMB was titrated with increasing amounts of nucleic acid, all four forms of nucleic acid caused strong polarisation of fluorescence, demonstrating the association with PHMB. The intensity and broad-spectrum binding of PHMB to all forms of nucleic acid has significant implications for the mechanism of action of this biocide.     

A 340 kDa hyaluronic acid secreted by human vascular smooth muscle cells regulates their proliferation and migration

The formation of atherosclerotic lesions is characterized by invasion of vascular smooth muscle cells (VSMC) into the tunica intima of the arterial wall and subsequently by increased proliferation of VSMC, a process apparently restricted to the intimal layer of blood vessels. Both events are preceded by the pathological overexpression of several growth factors, such as platelet-derived growth factor (PDGF) which is a potent mitogen for VSMC and can induce their chemotaxis. PDGF is generally not expressed in the normal artery but it is upregulated in atherosclerotic lesions. We have previously shown that PDGF-BB specifically stimulates proliferating VSMC to secrete a 340 kDa hyaluronic acid (HA-340). Here, we present evidence regarding the biological functions of this glycan. We observed that HA-340 inhibited the PDGF-induced proliferation of human VSMC in a dose-dependent manner and enhanced the PDGF-dependent invasion of VSMC through a basement membrane barrier. These effects were abolished following treatment of HA-340 with hyaluronidase. The effect of HA-340 on the PDGF-dependent invasion of VSMC coincided with increased secretion of the 72-kDa type IV collagenase by VSMC and was completely blocked by GM6001, a hydroxamic acid inhibitor of matrix metalloproteinases. HA-340 did not exert any chemotactic potency, nor did it affect chemotaxis of VSMC along a PDGF gradient. In human atheromatic aortas, we found that HA- 340 is expressed with a negative concentration gradient from the tunica media to the tunica intima and the atheromatic plaque. Our findings suggest that HA-340 may be linked to the pathogenesis of atherosclerosis, by modulating VSMC proliferation and invasion.      

Singular over-representation of an octameric palindrome, HIP1, in DNA from many cyanobacteria.

An octameric palindrome (5'-GCGATCGC-3') is abundant in cyanobacterial sequences within databases (GenBank/EMBL) and was designated HIP1 (highly iterated palindrome). The frequency of occurrence of all 256 octameric palindromes has now been determined in sub-databases revealing large and unique over-representation of HIP1 in cyanobacterial entries. DNA sequences from other bacteria were searched for any over-represented octameric palindromes analogous to HIP1. Only two sequences were identified, in the genomes of a thermophile and halophilic archaebacteria, although these were less abundant than HIP1 in cyanobacteria and relate to codon usage. To test the proposed widespread distribution of HIP1 in DNA from the cyanobacterium Synechococcus PCC 6301, randomly selected genomic clones were partly sequenced. HIP1 constituted 2.5% of the novel sequences, equivalent to a site on average once every 320 nucleotides. An oligonucleotide including HIP1 was also tested in PCR. Multiple products were obtained using template DNA from cyanobacterial strains in which HIP1 is abundant in known sequences, and some strains generated characteristic HIP-PCR banding patterns. However, analysis of DNA from one strain (not previously represented in databases) by random sequencing, HIP-PCR and Pvul digestion, confirms that not all cyanobacterial genomes are rich in HIP1.     

Protein evolution in different cellular environments: cytochrome b in sharks and mammals

DNA sequences for the mitochondrial cytochrome b gene were determined for 13 species of sharks. Rates and patterns of amino acid replacement are compared for sharks and mammals. Absolute rates of cytochrome b evolution are six times slower in sharks than in mammals. Bivariate plots of the number of nonsynonymous and silent transversions are indistinguishable in the two groups, however, suggesting that the differences in amino acid replacement rates are due primarily to differences in DNA substitution rates. Patterns of amino acid replacement are also similar in the two groups. Conserved and variable regions occur in the same parts of the cytochrome b gene, and there is little evidence that the types of amino acid changes are significantly different between the groups. Similarity in the relative rates and patterns of protein change between the two groups prevails despite dramatic differences in the cellular environments of sharks and mammals. Poor penetrance of physiological differences through to rates of protein evolution provides support for the neutral theory and suggests that, for cytochrome b, patterns of evolution have been relatively constant throughout much of vertebrate history.      

Distribution of the molossinus allele of Sry, the testis-determining gene, in wild mice

When the Y chromosome of the laboratory inbred mouse strain C57BL/6 (B6) is replaced by the Y of certain strains of Mus musculus domesticus, testis determination fails and all XY fetuses develop either as hermaphrodites or XY females (XY sex reversal). This suggests the presence of at least two alleles of Sry, the male-determining gene on the Y:M. m. domesticus and B6. The B6 Y chromosome is derived from the Japanese house mouse, M. m. molossinus and therefore carries a molossinus Sry allele. As a first step to determine how the molossinus Sry allele evolved, its distribution pattern was determined in wild mice. The cumulative data of 96 M. musculus samples obtained from 58 geographical locations in Europe, North Africa, and Asia show the molossinus Sry allele is restricted to Japan and the neighboring Asian mainland and confirm that Japanese M. m. molossinus mice were derived in part from a race of M. m. musculus from Korea or Manchuria. Sry polymorphisms, as illustrated by the molossinus Sry allele, can serve as molecular markers for studies on the evolution of wild M. musculus populations and can help determine the role sex determination plays in speciation.      

The biodegradation of tributyl phosphate by naturally occurring microbial isolates

The biodegradation of tributyl phosphate by a mixed culture of Pseudomonads was demonstrated. Growth and the rate of tributyl phosphate consumption were variable and divisible into rapid and slow rates. Rapidly growing, rapidly tributyl phosphate-utilising cultures contained a 22–24 kb DNA fragment isolated by two methods, which was not visible in the cultures growing slowly. The mixed culture gave five periods of rapid growth interspersed with periods of poor growth during 7 months of weekly subculture, with the 22–24 kb DNA fragment detectable during the rapidly growing periods only. Seven Pseudomonads isolated from the culture grew at the expense of tributyl phosphate as the sole phosphorus source but spontaneously and irreversibly lost this ability after eight serial subcultures.