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Summary The Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL) ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. Here, we demonstrate that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, we show that this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system., Graphical Abstract, Highlights • Notch ligands contain an N-terminal C2 phospholipid recognition domain • Ca2+-dependent lipid binding by Jagged 1 is required for optimal Notch activation • Lipid binding by Notch ligands is calcium dependent • Notch ligands require bound calcium at the N terminus for full activity, Handford, Lea, and colleagues now discover that the N-terminal portion of Notch ligands consists of a functional, calcium-dependent phospholipid recognition domain, the C2 domain. They also show that Notch-driven activation requires C2 domain activity in addition to canonical Notch-ligand binding.

To investigate the expression level and analyze the clinical significance of NT5C2, which is an nucleoside analogues metabolism related gene, in children with acute leukemia (AL).Real-time PCR and immunohistochemistry were presented to detect the level of NT5C2 mRNA and its protein product cN- Ⅱ in bone marrow samples of 63 patients initially diagnosed with AL, 15 patients who achieved complete remission, 7 patients who relapsed and 16 non- hematologic malignancie controls. The expression of NT5C2 mRNA in different groups of AL and its relevance with clinical indicators were analyzed.①The expression of NT5C2 mRNA in newly diagnosed B-ALL, TALL, AML and controls were 1.16 (0.89-2.25, 0.96 (0.74-1.25, 1.66 (0.84-3.15) and 0.88 (0.61-1.21), respectively. NT5C2 mRNA expression in AML (P0.01) and B-ALL (P0.05) cases were higher than that in controls; NT5C2 mRNA expression in T- ALL and in controls showed no significant difference (P0.05). Changes of NT5C2 mRNA level were observed between preliminary diagnosis and complete remission in 15 patients. NT5C2 mRNA levels were significantly decreased in complete remission stage than that in newly diagnosis AL (P0.01). NT5C2 mRNA levels of relapsed-refractory group were higher than that of complete remission group and controls (P0.01). ② Immunohistochemical staining results revealed that NT5C2 protein levels were consistent with the trend of mRNA levels. ③NT5C2 mRNA levels in AML (r=0.434) and T-ALL (r=0.389) were positively correlated with risk classification (P0.05). ④ During chemotherapy of patients with AML, the NR rate of bone marrow in NT5C2 high expression group was higher than that of low expression group after 9 days induction chemotherapy (35.2% vs 0) and before consolidation therapy (25.0% vs 0); The positive rate of minimal-residual disease (36.4% vs 14.3%) and relapse rate of AL (38.5% vs 28.6%) were increased in NT5C2 high expressed patients than that in low expressed patients, but all the differences were insignificant (P0.05).High expression of NT5C2 was found to be a related risk factor of AL children with unfavourable prognosis. NT5C2 promises a new target for guiding individualized chemotherapy and evaluating the prognosis of childhood acute leukemia and monitoring recurrence.

All-trans retinoic acid (ATRA) induces complete remission in almost all patients with acute promyelocytic leukemia (APL) via its ability to induce the in vivo differentiation of APL blasts. However, prolonged ATRA treatment can result in drug resistance. In previous studies, we generated a multi-drug-resistant HL60/ATRA cell line and found it to contain a new drug resistance-related gene segment, HA117. In this study, we demonstrate that ATRA induces multi-drug-resistant subpopulations of HL60 cells with a putative stem-like signature by up-regulating the expression of the new gene segment HA117. Western blot analysis and quantitative real-time PCR demonstrated that HA117 causes alternative splicing of regulator of G-protein signaling 6 (RGS6) and down-regulation of the expression of the GGL domain of RGS6, which plays an important role in DNA methyltransferase 1 (DNMT1) degradation. Moreover, DNMT1 expression was increased in multi-drug resistance HL60/ATRA cells. Knockdown of HA117 restored expression of the GGL domain and blocked DNMT1 expression. Moreover, resistant cells displayed a putative stem-like signature with increased expression of cancer steam cell markers CD133 and CD123. The stem cell marker, Nanog, was significantly up-regulated. In conclusion, our study shows that HA117 potentially promotes the stem-like signature of the HL60/ATRA cell line by inhibiting by the ubiquitination and degradation of DNMT1 and by down-regulating the expression of the GGL domain of RGS6. These results throw light on the cellular events associated with the ATRA-induced multi-drug resistance phenotype in acute leukemia.

Hirschsprung's disease (HSCR), the most common congenital malformation of the gut, is regulated by multiple signal transduction pathways. Several components of these pathways are important targets for microRNAs (miRNAs). Multiple miRNAs have been associated with the pathophysiology of HSCR, and serum miRNAs profiles of HSCR patients have been reported, but miRNA expression in HSCR colon tissue is almost completely unexplored. Using microarray technology, we screened colon tissue to detect miRNAs whose expression profiles were altered in HSCR and identify targets of differentially expressed miRNAs. Following filtering of low-intensity signals, data normalization, and volcano plot filtering, we identified 168 differentially expressed miRNAs (104 up-regulated and 64 down-regulated). Fifty of these mRNAs represent major targets of dysegulated miRNAs and may thus important roles in the pathophysiology of HSCR. Pathway analysis revealed that 7 of the miRNA targets encode proteins involved in regulation of cell proliferation and migration via RET and related signaling pathways (MAPK and PI3K/AKT). Our results identify miRNAs that play key roles in the pathophysiology of the complex multi-factorial disease HSCR.