p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients

Membrane attack complex inhibitor CD59a protects against focal cerebral ischemia in mice

<p>Abstract</p> <p>Background</p> <p>The complement system is a crucial mediator of inflammation and cell lysis after cerebral ischemia. However, there is little information about the exact contribution of the membrane attack complex (MAC) and its inhibitor-protein CD59.</p> <p>Methods</p> <p>Transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in young male and female CD59a knockout and wild-type mice. Two models of MCAO were applied: 60 min MCAO and 48 h reperfusion, as well as 30 min MCAO and 72 h reperfusion. CD59a knockout animals were compared to wild-type animals in terms of infarct size, edema, neurological deficit, and cell death.</p> <p>Results and Discussion</p> <p>CD59a-deficiency in male mice caused significantly increased infarct volumes and brain swelling when compared to wild-type mice at 72 h after 30 min-occlusion time, whereas no significant difference was observed after 1 h-MCAO. Moreover, CD59a-deficient mice had impaired neurological function when compared to wild-type mice after 30 min MCAO.</p> <p>Conclusion</p> <p>We conclude that CD59a protects against ischemic brain damage, but depending on the gender and the stroke model used.</p

Constraining the nuclear equation of state at subsaturation densities

Only one third of the nucleons in $^{208}$Pb occupy the saturation density area. Consequently nuclear observables related to average properties of nuclei, such as masses or radii, constrain the equation of state (EOS) not at saturation density but rather around the so-called crossing density, localised close to the mean value of the density of nuclei: $\rho\simeq$0.11 fm$^{-3}$. This provides an explanation for the empirical fact that several EOS quantities calculated with various functionals cross at a density significantly lower than the saturation one. The third derivative M of the energy at the crossing density is constrained by the giant monopole resonance (GMR) measurements in an isotopic chain rather than the incompressibility at saturation density. The GMR measurements provide M=1110 $\pm$ 70 MeV (6% uncertainty), whose extrapolation gives K$_\infty$=230 $\pm$ 40 MeV (17% uncertainty).Comment: 4 pages, 4 figure

CATMA: a complete Arabidopsis GST database

The Complete Arabidopsis Transcriptome Micro Array (CATMA) database contains gene sequence tag (GST) and gene model sequences for over 70% of the predicted genes in the Arabidopsis thaliana genome as well as primer sequences for GST amplification and a wide range of supplementary information. All CATMA GST sequences are specific to the gene for which they were designed, and all gene models were predicted from a complete reannotation of the genome using uniform parameters. The database is searchable by sequence name, sequence homology or direct SQL query, and is available through the CATMA website at http://www.catma.or

Functional analysis and identification of cis-regulatory elements of human chromosome 21 gene promoters

Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to ∼500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulatio

Functional analysis and identification of cis-regulatory elements of human chromosome 21 gene promoters

Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum. We observed high correlations between promoter activities and endogenous transcript levels, RNA polymerase II occupancy, CpG islands and core promoter elements. Truncation of a subset of 62 promoters to ∼500 bp revealed that truncation rarely resulted in loss of activity, but rather in loss of responses to external stimuli, suggesting the presence of cis-regulatory response elements within distal promoter regions. In these regions, we found a strong enrichment of transcription factor binding sites that could potentially activate gene expression in the presence of stimuli. This study illustrates the modular functional architecture of chromosome 21 promoters and helps to reveal the complex mechanisms governing transcriptional regulation

Using hippocampal microRNA expression differences between mouse inbred strains to characterise miRNA function

Micro-RNAs (miRNAs) are short, single-stranded, noncoding RNAs that are involved in the regulation of protein-coding genes at the level of messenger RNA (mRNA). They are involved in the regulation of numerous traits, including developmental timing, apoptosis, immune function, and neuronal development. To better understand how the expression of the miRNAs themselves is regulated, we looked for miRNA expression differences among four mouse inbred strains, A/J, BALB/cJ, C57BL/6J, and DBA/2J, in one tissue, the hippocampus. A total of 166 miRNA RT-PCR assays were used to screen RNA pools for each strain. Twenty miRNA species that were markedly different between strains were further investigated using eight individual samples per strain, and 11 miRNAs showed significant differences across strains (p < 0.05). This is the first observation of miRNA expression differences across inbred mice strains. We conducted an in silico correlation analysis of the expression of these differentially expressed miRNAs with phenotype data and mRNA expression to better characterise the effects of these miRNAs on both phenotype and the regulation of mRNA expression. This approach has allowed us to nominate miRNAs that have potential roles in anxiety, exploration, and learning and memory

Molecular characterisation of spreading depression-induced preconditioning

Preconditioning refers to the adaptative cytoprotection that can be induced by a variety of sublethal insults (e.g. a short period of hypoxia) and which increases the brain resistance to a subsequent, potentially lethal insult (e.g. severe ischemia). This fundamental biological process provides an alternative scientific rationale and strategy for the discovery of effective neuroprotective strategies. Our objective is to identify the biological determinants of increased tolerance through the determination of changes in gene expression and protein profiles at several time points after induction of preconditioning. We are focusing on preconditioning induced by repetitive cortical spreading depression (CSD), because this stimulus allows the preparation of large samples of mouse cortex that are preconditioned evenly and consistently. Differential genomics at time points 1, 3 and 6 h post CSD revealed significant changes in the expression of a large number of genes, among which 178 possible candidates had their expression changed by > 100 folds during this period. A high degree of correlation was found between the 3 time points, indicating good experimental reproducibility, but a further evaluation of the candidate genes is still required. Only a few selected proteins were examined by Western blot analysis, 24 h after CSD. New findings include significant changes in immunoreactivity for the glutamate AMPA receptor GluR1 and GluR2 subunits, and especially a 15-fold increase in that of the a7 nicotinic acetylcholine receptor (a7 nAChR) subunit