The aim of present study was to evaluate CD4(+) /CD8(+) ratio and CD4(+) CD25(hi) FoxP3(+) Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4(+) /CD8(+) ratio and Tregs in 82 patients and 50 controls. CD8(+) T-cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8(+) T-cell counts compared with stable GV patients (p = 0.001). The CD4(+) /CD8(+) ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4(+) /CD8(+) ratio and natural Tregs in frequency and function might be involved in the T-cell mediated pathogenesis of GV and its progression.

Limiting the levels of homologous recombination (HR) that occur at sites of DNA damage is a major role of BLM helicase. However, very little is known about the mechanisms dictating its relocalization to these sites. Here, we demonstrate that the ubiquitin/SUMO-dependent DNA damage response (UbS-DDR), controlled by the E3 ligases RNF8/RNF168, triggers BLM recruitment to sites of replication fork stalling via ubiquitylation in the N-terminal region of BLM and subsequent BLM binding to the ubiquitin-interacting motifs of RAP80. Furthermore, we show that this mechanism of BLM relocalization is essential for BLM's ability to suppress excessive/uncontrolled HR at stalled replication forks. Unexpectedly, we also uncovered a requirement for RNF8-dependent ubiquitylation of BLM and PML for maintaining the integrity of PML-associated nuclear bodies and as a consequence the localization of BLM to these structures. Lastly, we identified a novel role for RAP80 in preventing proteasomal degradation of BLM in unstressed cells. Taken together, these data highlight an important biochemical link between the UbS-DDR and BLM-dependent pathways involved in maintaining genome stability.

BLM helicase, the protein mutated in Bloom syndrome, is involved in signal transduction cascades after DNA damage. BLM is phosphorylated on multiple residues by different kinases either after stress induction or during mitosis. Here, we have provided evidence that both Chk1 and Chk2 phosphorylated the NH 2 -terminal 660 amino acids of BLM. An internal region within the DExH motif of BLM negatively regulated the Chk1/Chk2-dependent NH 2 -terminal phosphorylation event. Using in silico analysis involving the Chk1 structure and its known substrate specificity, we predicted that Chk1 should preferentially phosphorylate BLM on serine 646 (Ser 646 ). The prediction was validated in vitro by phosphopeptide analysis on BLM mutants and in vivo by usage of a newly generated phosphospecific polyclonal antibody. We showed that the phosphorylation at Ser 646 on BLM was constitutive and decreased rapidly after exposure to DNA damage. This resulted in the diminished interaction of BLM with nucleolin and PML isoforms, and consequently decreased BLM accumulation in the nucleolus and PML nuclear bodies. Instead, BLM relocalized to the sites of DNA damage and bound with the damage sensor protein, Nbs1. Mutant analysis confirmed that the binding to nucleolin and PML isoforms required Ser 646 phosphorylation. These results indicated that Chk1-mediated phosphorylation on BLM at Ser 646 might be a determinant for regulating subnuclear localization and could act as a marker for the activation status of BLM in response to DNA damage. Mol Cancer Res; 8(9); 1234-47. ©2010 AACR.

During development, the heart growths through addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, wilms tumor 1 transcription factor a (wt1a) and b (wt1b) are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a/b expression is maintained in proepicardial cells, it is downregulated in those pericardial cells contributing to cardiomyocytes from the developing heart. Sustained wt1 expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a/b-expressing cardiomyocytes changed their cell adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1 acts as a break for cardiomyocyte differentiation and ectopic wt1 expression in cardiomyocytes can lead to their transdifferentiation into epicardial like cells.

Zebrafish have the capacity to fully regenerate the heart after an injury, which lies in sharp contrast to the irreversible loss of cardiomyocytes after a myocardial infarction in humans. Transcriptomics analysis has contributed to dissect underlying signaling pathways and gene regulatory networks in the zebrafish heart regeneration process. This process has been studied in response to different types of injuries namely: ventricular resection, ventricular cryoinjury, and genetic ablation of cardiomyocytes. However, there exists no database to compare injury specific and core cardiac regeneration responses. Here, we present a meta-analysis of transcriptomic data of regenerating zebrafish hearts in response to these three injury models at 7 days post injury (7dpi). We reanalyzed 36 samples and analyzed the differentially expressed genes (DEG) followed by downstream Gene Ontology Biological Processes (GO:BP) analysis. We found that the three injury models share a common core of DEG encompassing genes involved in cell proliferation, the Wnt signaling pathway and genes that are enriched in fibroblasts. We also found injury-specific gene signatures for resection and genetic ablation, and to a lower extent the cryoinjury model. Finally, we present our data in a user-friendly web interface that displays gene expression signatures across different injury types and highlights the importance to consider injury-specific gene regulatory networks when interpreting the results related to cardiac regeneration in the zebrafish. The analysis is freely available at: https://mybinder.org/v2/gh/MercaderLabAnatomy/PUB_Botos_et_al_2022_shinyapp_binder/HEAD?urlpath=shiny/bus-dashboard/.