Transcriptional Mutagenesis Induced by 8-Oxoguanine in Mammalian Cells

Most of the somatic cells of adult metazoans, including mammals, do not undergo continuous cycles of replication. Instead, they are quiescent and devote most of their metabolic activity to gene expression. The mutagenic consequences of exposure to DNA–damaging agents are well documented, but less is known about the impact of DNA lesions on transcription. To investigate this impact, we developed a luciferase-based expression system. This system consists of two types of construct composed of a DNA template containing an 8-oxoguanine, paired either with a thymine or a cytosine, placed at defined positions along the transcribed strand of the reporter gene. Analyses of luciferase gene expression from the two types of construct showed that efficient but error-prone transcriptional bypass of 8-oxoguanine occurred in vivo, and that this lesion was not repaired by the transcription-coupled repair machinery in mammalian cells. The analysis of luciferase activity expressed from 8OG:T-containing constructs indicated that the magnitude of erroneous transcription events involving 8-oxoguanine depended on the sequence contexts surrounding the lesion. Additionally, sequencing of the transcript population expressed from these constructs showed that RNA polymerase II mostly inserted an adenine opposite to 8-oxoguanine. Analysis of luciferase expression from 8OG:C-containing constructs showed that the generated aberrant mRNAs led to the production of mutant proteins with the potential to induce a long-term phenotypical change. These findings reveal that erroneous transcription over DNA lesions may induce phenotypical changes with the potential to alter the fate of non-replicating cells

Mapping of HNF4α target genes in intestinal epithelial cells

© 2009 Boyd et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

The CSF-1 receptor fashions the intestinal stem cell niche

AbstractGastrointestinal (GI) homeostasis requires the action of multiple pathways. There is some controversy regarding whether small intestine (SI) Paneth cells (PCs) play a central role in orchestrating crypt architecture and their relationship with Lgr5+ve stem cells. Nevertheless, we previously showed that germline CSF-1 receptor (Csf1r) knock out (KO) or Csf1 mutation is associated with an absence of mature PC, reduced crypt proliferation and lowered stem cell gene, Lgr5 expression. Here we show the additional loss of CD24, Bmi1 and Olfm4 expression in the KO crypts and a high resolution 3D localization of CSF-1R mainly to PC. The induction of GI-specific Csf1r deletion in young adult mice also led to PC loss over a period of weeks, in accord with the anticipated long life span of PC, changed distribution of proliferating cells and this was with a commensurate loss of Lgr5 and other stem cell marker gene expression. By culturing SI organoids, we further show that the Csf1r−/− defect in PC production is intrinsic to epithelial cells as well as definitively affecting stem cell activity. These results show that CSF-1R directly supports PC maturation and that in turn PCs fashion the intestinal stem cell niche

Lawsonia intracellularis exploits β-catenin/Wnt and Notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation

Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE

Etching of low-k materials in high density fluorocarbon plasma

Low dielectric constant materials (low-k) are used as interlevel dielectrics in integrated circuits. This paper concerns the etching process of these materials in high density plasma with the aim to provide some insights concerning the etch mechanisms. Materials studied are methylsilsesquioxane (MSQ) polymers, either dense (SiOC) or containing 40% of porosity (porous SiOC). Amorphous hydrogenated silicon carbide (SiC) material, used as hard mask and/or etch stop layer, is also investigated. Etch is performed in an inductively coupled reactor using fluorocarbon gases, which have proven to be very successful in the etch of conventional SiO2. First, etching with hexafluoroethane (C2F$_{6})$ is performed. Although etch rates are high, etch selectivities with respect to SiC are weak. So, oxygen, argon, and hydrogen are added to C2F6 with the aim of improving selectivities. The best selectivity is obtained for the C2F6/H2 (10%–90%) mixture. To understand etch rate and selectivity variations, plasma analyses by optical emission spectroscopy are correlated to surface analysis using X-Ray Photoelectron Spectroscopy (XPS). In general, atomic fluorine concentration in the plasma explains the etch rate, while the presence of a fluorocarbon layer on the surface is well correlated to the selectivity. To ensure that the etch process does not affect materials properties, and particularly their dielectric constant, bulk analysis by Fourier Transformed Infra-Red spectroscopy and images by Scanning Electron Microscopy have also been carried out

Characterizing fish communities associated with drifting fish aggregating devices (FADs) in the Western Indian Ocean using underwater visual surveys [+ Erratum paru dans Aquat. Living Resour. 2017, 30, 26]

We adapted a visual census method, mainly used in demersal and reef fish studies, to characterize fish communities associated to drifting fish aggregating devices (FADs) in the Western Indian Ocean. Drifting FAD associated fishes from both equatorial (Seychelles) and tropical waters (Reunion Island) were examined by divers. A total of 32 species (belonging to 16 families) were observed associated with drifting FADs in equatorial waters, and 24 species (14 families) were found around FADs in tropical waters. Twenty species were found in both regions. The highest number of species observed at a single FAD was 18 (12 +/- 2, mean +/- SD) in equatorial and 13 (10 +/- 3) in tropical waters, not counting circumnatant species loosely associated with the FAD. Some species like Kyphosus vaigiensis, Canthidermis maculata, Elagatis bipinnulata, Acanthocybium solandri and Coryphaena hippurus were observed on all or most of the surveys. In this study, the contribution in biomass of the 18 common species associated with drifting FADs (but excluding circumnatant species), represents more than 98% of the biomass. The overall biomass values of closely associated species remains well below tuna biomass estimates for circumnatant tuna schools at FADs, estimated as high as 200 tons. The species that most significantly contribute to the by-catch in tuna purse-seines logically match those that showing the highest biomass values in our surveys (Carcharhinus spp., Elagatis bipinnulata, Coryphaena hippurus, Canthidermis maculata, and Acanthocybium solandri). One of the most abundant and ubiquitous species in our study was the spotted oceanic triggerfish Canthidermis maculata that sometimes formed massive schools of many thousands individuals around the drifting FADs. Future research is needed to explore the role of such non tuna species in the attraction and aggregation processes of tuna around drifting FADs