Phosphorylation of liver X receptor alpha selectively regulates target gene expression in macrophages

Dysregulation of liver X receptor alpha (LXRalpha) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRalpha target gene selectivity is achieved by modulation of LXRalpha phosphorylation. Under basal conditions, LXRalpha is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRalpha S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRalpha S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRalpha-responsive genes.     

The influence of sodium metavanadate on the process of diabetogenesis in BB rats

Vanadium has been shown to be beneficial in the oral treatment of animal models of type 1 and type 2 diabetes. The aim of the study was to evaluate the short-term effects of sodium metavanadate in prediabetic BB-DP rats. To do this, 96 rats were divided into 4 equal groups. Groups VI, V2, V3 were treated with sodium metavanadate (0.1, 0.2 and 0.3 mg/ml respectively) and sodium chloride (0.5 mg/ml) in drinking water for 7 days. Group C received only sodium chloride (0.5 mg/ml). Blood glucose (BG), glycosuria, ketonuria, body weight and insulinemia were determined. The age of onset of diabetes was significantly higher for groups V2, V3 compared to group C, (p < 0.05) and depends on the metavanadate concentration (V3 vs. V1, p=0.006). The incidence of diabetes was lower in the rats treated with metavanadate than in the control group, but this difference was not statistically significant. In diabetic rats, the BG at the onset was higher in group C than in groups V, p < 0.05. Insulinemia, at the onset of the treatment as well as immediately after its cessation showed a drop in the treatment groups, proportionally to the dosage of vanadium, but later increased slowly and continuously until the end of the experiment. In conclusion, metavanadate delays the development of diabetes in BB-DP rats, but does not prevent its onset. A milder form of diabetes occurs in diabetic rats treated with metavanadate. The effects depend on the metavanadate concentration and 0.2 mg/ml is preferable.     

Maintenance of paternal methylation and repression of the imprinted H19 gene requires MBD3

Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.     

YOC,A new strategy for pairwise alignment of collinear genomes

Background

Comparing and aligning genomes is a key step in analyzing closely related genomes. Despite the development of many genome aligners in the last 15 years, the problem is not yet fully resolved, even when aligning closely related bacterial genomes of the same species. In addition, no procedures are available to assess the quality of genome alignments or to compare genome aligners.

Results

We designed an original method for pairwise genome alignment, named YOC, which employs a highly sensitive similarity detection method together with a recent collinear chaining strategy that allows overlaps. YOC improves the reliability of collinear genome alignments, while preserving or even improving sensitivity. We also propose an original qualitative evaluation criterion for measuring the relevance of genome alignments. We used this criterion to compare and benchmark YOC with five recent genome aligners on large bacterial genome datasets, and showed it is suitable for identifying the specificities and the potential flaws of their underlying strategies.

Conclusions

The YOC prototype is available at https://github.com/ruricaru/YOC. It has several advantages over existing genome aligners: (1) it is based on a simplified two phase alignment strategy, (2) it is easy to parameterize, (3) it produces reliable genome alignments, which are easier to analyze and to use.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0530-3) contains supplementary material, which is available to authorized users.     

Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes

Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.     

Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L‐T3, L‐T4, and D‐T4

A fast screening method of whole blood was proposed for enantiorecognition of free L‐T₃ , L‐T₄, and D‐T₄. Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L‐T₃ , L‐T₄, and D‐T₄. For the enantiorecognition of free L‐T₄ and D‐T₄ in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L‐T₃ (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L‐T4, and D‐T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP‐based microsensor. For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). Free L‐T₃ , L‐T₄, and D‐T₄ were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%). Chirality 27:973–978, 2015. © 2015 Wiley Periodicals, Inc.