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Pharmacology of Withanolide A
Published in Amritpal Singh Saroya, Contemporary Phytomedicines, 2017
Exposure of MDA-MB-231 and MCF-7 human breast cancer cells to pharmacological concentrations of withanolide-A resulted in cleavage (activation) of Notch 2 as well as Notch 4, which was accompanied by 220 Contemporary Phytomedicines transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays.
Molecular diagnosis of endometriosis
Published in Carlos Simón, Linda C. Giudice, The Endometrial Factor, 2017
Lusine Aghajanova, Linda C. Giudice
Recombination signal binding protein for immunoglobulin kappa J region (RBPJ), a DNA binding protein that interacts with the Notch intracellular domain to activate transcription of genes that inhibit cell differentiation, is increased in endometrium from women with endometriosis (60,61). Notch signaling is essential for endometrial stromal differentiation in the mouse, baboon, and human (62,63), and interestingly, decreased expression of Notch signaling in endometrium from women and baboons with endometriosis, as well as isolated eSFs, has been observed and is associated with impaired decidualization through the downregulation of FOXO1A (64).
Normal fetal skeletal growth and development
Published in Christine M Hall, Amaka C Offiah, Francesca Forzano, Mario Lituania, Michelle Fink, Deborah Krakow, Fetal and Perinatal Skeletal Dysplasias, 2012
Christine M Hall, Amaka C Offiah, Francesca Forzano, Mario Lituania, Michelle Fink, Deborah Krakow
The segmentation process is genetically controlled by an oscillating ‘segmentation clock’, which is determined by pulses of signalling of Notch, Wnt and fibroblast growth factor (FGF). The Notch pathway is particularly important for proper patterning of the developmental axes and vertebral modelling and many components of this pathway have been identified (DLL1, DLL3, LFNG, MIB1, POFUT1, PSEN1, CSL/RBPJ), as well as some of the target genes (HES7, MESP2, LFNG).
Recent advances in high-throughput flow cytometry for drug discovery
Published in Expert Opinion on Drug Discovery, 2021
In addition to immuno-oncology applications, HTFC has also been used to screen over 80,000 compounds in a search for inhibitors of nonhomologous end joining (NHEJ)-mediated DNA repair. This screen used a custom Cluster Cytometry platform to enable sampling from 4 x 384-well plates simultaneously. To further simplify the protocol, forward and side scatter were used to identify proliferation and cell viability, respectively, and a GFP readout used to measure NHEJ, rather than additional dye or antibody labeling steps [102]. Further investigation of the DNA damage response can be performed by using γH2AX as a marker of double-strand breaks and MRE11 as a marker of double-strand break repair, and has been used to show that non-responders to PARP inhibitors have significantly higher ratio of γH2AX/MRE11 compared to those who did respond [103]. A recent paper describes an HTFC assay using glutathione-coated beads to capture GST-RBPJ fusion protein, and fluorescent DNA containing RBPJ binding motif as a tracer to screen inhibitors of RBPJ-DNA interaction. This screen together with a follow-up cell assay has identified auranofin that disrupts RBPJ-DNA interaction and downregulates Notch-dependent transcription activation that is involved in cancer cell initiation and proliferation [104]. Flow cytometers are also uniquely positioned to analyze exosomes, and HTFCs such as the MACSQuant have been used to quantify exosome release in prostate cancer cell models following compound treatment [105].
Current opinion in the molecular genetics of Adams-Oliver syndrome
Published in Expert Opinion on Orphan Drugs, 2019
The highly conserved Notch pathway is known to be critical for many developmental processes. The canonical Notch cascade is activated by extracellular binding of a delta-like (DLL) or jagged (JAG) ligand from an adjacent cell, triggering cleavage of the Notch intracellular domain (NICD). NICD subsequently translocates to the nucleus, where it complexes with the transcription factor RBPJ to regulate the expression of Notch target genes, for example HES1 and HEY1. In AOS, missense mutations within the DNA-binding domain of RBPJ have been shown to attenuate its binding to the HES1 promoter, leading to dysregulated Notch signaling [14]. Similarly, NOTCH1 mutation-positive patients have reduced NOTCH1 expression with an associated decrease in transcript abundance of HES1 and HEY1 [7]. Of interest, ARHGAP31 has recently been discovered to exhibit transcriptional activity, which is absent in the recurrent p.GlnQ683* nonsense variant, leading to the possibility of potential cross talk between ARHGAP31 and the Notch signaling pathway [28].
Advances in the creation of animal models of paroxysmal nocturnal hemoglobinuria
Published in Hematology, 2021
Immune escape and anti-apoptotic mechanisms can explain the development and resistance of mutated PNH cells yet are not sufficient to describe the proliferation of PNH clones. Therefore, the possible effects of secondary mutated genes, other than PIG-A, have attracted much attention. Globally, researchers have used whole gene sequencing technology to screen for possible secondary mutated genes [7]. Our research center conducted a full exon sequencing with 13 PNH patients and found multiple secondary mutated genes including RBPJ, MUC4, SUZ12, MAGEC1, CUX1, MLL2, TET2, and CEBP2 [8]. Further in vitro tests revealed that the expression level of RBPJ was significantly higher in the CD59- than in CD59+ cells of PNH patients. When siRNA-RBPJ was used to inhibit the expression of RBPJ in PNH primary cells, their apoptosis rate increased, their proliferation activity decreased with the extension of transfection time, and the cells were blocked in the G0/G1 phase [9]. However, due to the absence of adequate in vivo verification methods, the precise pathogenic gene and its mechanism of action has not been determined. This therefore necessitates the establishment of a PNH animal model to study the pathogenesis of the disease. Several domestic and foreign scholars have developed PNH animal models through PIG-A gene editing, but some typical clinical manifestations such as hemolysis have not been observed, thereby necessitating an improvement of the models. The successful establishment of a clinically relevant PNH animal model will be of great significance to explore the pathogenesis of the disease and identify new therapeutic targets.