Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His6-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15-His6. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, EGF14-15-His6 was efficiently released from the escorting tag. EGF14-15-His6 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15-His6. Hence, a large amount of EGF14-15-His6 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.

Background: Exposure to inorganic arsenic (iAs) remains a global public health problem. The liver is the main target organ of arsenic, leading to arsenic-induced liver fibrosis. Autophagy is involved. Phosphatase and tensin homology deleted on chromosome ten (PTEN) may participate in arsenic-induced liver fibrosis by regulating autophagy, but the exact mechanisms remain unclear. We established a mouse model of arsenic poisoning through the drinking water, and a fibrosis model using the huma stellate cell (HSC) line LX-2, which was exposed to NaAsO₂ for 24h. HE and Masson staining was adopted to observe the degree of liver fibrosis. The cells were transfected using PTEN overexpression plasmid. Western blot and qRT-PCR were used to determine the levels of protein/mRNA expression. The in vivo results were confirmed in HSCs exposed to NaAsO₂, with changes suggesting fibrosis as seen in mice. NaAsO₂ upregulated the expression of the autophagic markers microtubule-associated protein light chain A/B (LC3), recombinant human autophagy effector protein (Beclin-1), hairy and enhancer of split homolog-1 (HES1), but downregulated PTEN. α-smooth muscle actin (α-SMA) expression was significantly upregulated in all NaAsO₂ groups. PTEN overexpression altered NaAsO₂-induced autophagy which LC3, Beclin-1 were downregulated. Notch1, HES1, α-SMA, and collagenⅠexpression were all downregulated in the NaAsO₂ groups. In conclusion, PTEN overexpression might decrease autophagy and inhibit fibrosis progression caused by this toxin. The NOTCH1/HES1 pathway is likely to be involved in this process. Most previous studies did not investigate PTEN and arsenic-induced liver fibrosis specifically. The present study highlights the importance of targeting PTEN for the management of arsenic exposure.

In the present work we present a methodology for teaching the basis of the genetic code through music composition, with the aim to combine science and arts learning. The project was carried out by 155 students, the so-called MARGA Consortium, with ages comprised between 10 and 17 years from different public schools located in the Principality of Asturias, Spain. The different groups generated 8 different music works using a short genetic sequence obtained from the human notch1 gene, receptor of mutations leading to chronic lymphocytic leukemia.

Hepatitis B virus (HBV) is the dominant risk factor for hepatocellular carcinoma (HCC). HBV X protein (HBx) plays crucial roles in HCC carcinogenesis. HBx interferes with several signaling pathways including Notch1 pathway in HCC. In our study, we found that Notch1 was highly expressed in HCC especially in large HCC. Notch1 and HBx co-localized in HCC and their levels were positively correlated with each other. Notch1 expression was more elevated in HepG2.2.15 than that in HepG2. HBx activated Notch1 pathway in HepG2.2.15. Repression of HBx and Notch1 pathway attenuated the growth of HepG2.2.15. Notch1, ERK and AKT pathways were inhibited after a γ-secretase inhibitor treatment. Dual-specificity phosphatase 1 (DUSP1) and phosphatase and tensin homolog (PTEN) were up-regulated after the γ-secretase inhibitor treatment and Hes1 inhibition. Luciferase reporter assays showed that Hes1 repressed the promoters of DUSP1 and PTEN and this was reverted by γ-secretase inhibitor treatment. Western blotting demonstrated that DUSP1 dephosphorylated pERK and PTEN dephosphorylated pAKT. Collectively, we reported a link among HBx, Notch1 pathway, DUSP1/PTEN, and ERK/AKT pathways, which influenced HCC cell survival and could be a therapeutic target for HCC.