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Background information. The DM (doublesex and male aberrant-3) genes implicated in sexual development in diverse metazoan organisms have been proved to be involved in development of non-gonadal tissues. The aim of the present study was to identify and characterize Dmrt3 (DM-related transcription factor 3) of zebrafish.Results. Zebrafish Dmrt3 has a conserved DMA domain, besides a common DM domain, which clustered it into the DMRTA subfamily. During embryogenesis, Dmrt3 expression increases gradually to a high level at pharyngula stage, which is restricted to the olfactory placode and the neural tube. In the juvenile zebrafish, the gene expression is first detected in undifferentiated gonad on 17 dpf (day post-fertilization). In adult, Dmrt3 is expressed only in the developing germ cells of both gonads, mainly in spermatogonia, spermatocytes and developing oocytes. The Dmrt3 has a functional NLS (nuclear localization signal) K41GHKR45within the DM domain, which ensures that Dmrt3 exerts its role in the nucleus. Moreover, EMSA (electrophoretic mobility-shift assay) indicates that the Dmrt3-derived DM polypeptide binds to similar sites of both targets of DSX (doublesex) and MAB-3 (male aberrant-3).Conclusion. These results suggest that as a DNA-binding protein, zebrafish Dmrt3 may function in the nucleus as a potential transcription factor to exert potential roles in the development of the olfactory placode, the neural tube and germ cells. [ABSTRACT FROM AUTHOR]

Summary Thymocytes expressing self-reactive T-cell receptors (TCR) are eliminated in the thymus through a TCR-mediated signal. This cell death signal (negative selection) generates nuclear morphological change and DNA fragmentation in thymocytes. However, the pathway leading to DNA fragmentation of thymocytes following TCR engagement remains obscure. In this study, we investigated the localization and function of caspase-activated DNAse (CAD) and its inhibitor (ICAD) in thymocytes prior to or after in vivo TCR stimulation. We showed that CAD and ICAD are co-localized in microsome, nuclei and cytosol in unstimulated thymocytes. Following in vivo TCR engagement, ICAD located in cytosol and microsome was degraded and the resulting activated CAD induced chromosomal DNA fragmentation. CAD present in cytosol and microsome of unstimulated thymocytes was activated by recombinant caspase-3, and microsomal CAD was released to the cytosol. These results demonstrate that TCR engagement of thymocytes induces caspase-3-dependent activation of CAD localized in both cytosol and microsome, leading to DNA fragmentation in harmony. [ABSTRACT FROM AUTHOR]

Introduction: Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal disease of hematopoietic stem cells. It is caused by somatic mutation of the X‐linked PIGA gene, resulting in a deficient expression of glycosylphosphatidylinositol‐anchored proteins (GPI‐APs). In this study, we aimed to explore the diagnostic value of next‐generation sequencing (NGS) and potential molecular basis in PNH patients. Methods: Genomic DNA of 85 PNH patients was analyzed by a 114‐gene NGS panel. Results: Mutational analysis of PIGA identified 124 mutations in 92% PNH patients, including 101 distinct mutations and 23 recurrent mutations. Among them, 102 mutations were newly reported. Most mutations were located in exon 2 of PIGA gene, and truncated mutation was the most common one. Other mutations were detected in 26 out of 85 cases, including five cases of DNMT3A variants, four cases of ASXL1 variants, and four cases of U2AF1 variants. Clonal analysis was performed in one case and outlined a linear evolution pattern in classic PNH. There was a positive correlation between number of PIGA mutations and fraction of GPI‐APs deficient granulocytes. Conclusion: The detection of PIGA mutations and additional variants by targeted NGS not only shed light on the genetic characteristics of PNH, but also provided an important reference value in the diagnosis of PNH at molecular level. [ABSTRACT FROM AUTHOR]