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Abstract Background l-ascorbic acid (Asc) plays a pivotal role in regulating various biological processes, including somatic cell reprogramming, through multiple pathways. However, it remains unclear whether Asc regulates reprogramming directly or functions through its metabolites. Results Asc exhibited dual capabilities in promoting reprogramming through both 2,3-diketo-l-gulonic acid (DKG), a key metabolite during Asc degradation, dependent and independent routes. On the one hand, Asc facilitated reprogramming by promoting cell proliferation and inducing the conversion from pre-induced pluripotent stem cells (pre-iPSCs) to iPSCs through DKG-independent pathways. Additionally, Asc triggered mesenchymal-epithelial transition (MET) and activated glycolysis via DKG-dependent mechanisms. Notably, DKG alone activated a non-canonical tricarboxylic acid cycle characterized by increased succinate, fumarate, and malate. Consequently, this shift redirected oxidative phosphorylation toward glycolysis and induced MET. Moreover, owing to its antioxidant capabilities, Asc directly inhibited glycolysis, thereby preventing positive feedback between glycolysis and epithelial-mesenchymal transition, ultimately resulting in a higher level of MET. Conclusion These findings unveil the intricate functions of Asc in the context of reprogramming. This study sheds light on the DKG-dependent and -independent activities of Asc during reprogramming, offering novel insights that may extend the application of Asc to other biological processes.

Abstract Mutation-induced malfunction of ten-eleven translocation methylcytosine dioxygenase 2 (TET2) is widely reported in haematological malignancies. However, the role of TET2 in solid cancers, including colorectal cancer (CRC), is unclear. Here, we found that TET2 malfunction in CRC is mostly due to decreased nuclear localization and that nuclear localization of TET2 is correlated with better survival of patients. To explore the underlying mechanisms, 14 immortalized solid tumour cell lines and 12 primary CRC cell lines were used. TET2 was mostly detected in the nucleus, and it induced significant DNA demethylation and suppressed cell growth by demethylating RORA and SPARC in cell lines like SW480. While in cell lines like SW620, TET2 was observed in the cytosol and did not affect DNA methylation or cell growth. Further examination with immunoprecipitation–mass spectrometry illustrated that β-catenin activation was indispensable for the nuclear localization and tumour suppression effects of TET2. In addition, the β-catenin pathway activator IM12 and the TET2 activator vitamin C were used simultaneously to enhance the effects of TET2 under low-expression conditions, and synergistic inhibitory effects on the growth of cancer were observed both in vitro and in vivo. Collectively, these data suggest that β-catenin-mediated nuclear localization of TET2 is an important therapeutic target for solid tumours.

Abstract Retinoic acid (RA) and 2-phospho-L-ascorbic acid trisodium salt (AscPNa) promote the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. In the current studies, the lower abilities of RA and AscPNa to promote reprogramming in the presence of each other suggested that they may share downstream pathways at least partially. The hypothesis was further supported by the RNA-seq analysis which demonstrated a high-level overlap between RA-activated and AscPNa activated genes during reprogramming. In addition, RA upregulated Glut1/3, facilitated the membrane transportation of dehydroascorbic acid, the oxidized form of L-ascorbic acid, and subsequently maintained intracellular L-ascorbic acid at higher level and for longer time. On the other hand, AscPNa facilitated the mesenchymal-epithelial transition during reprogramming, downregulated key mesenchymal transcriptional factors like Zeb1 and Twist1, subsequently suppressed the expression of Cyp26a1/b1 which mediates the metabolism of RA, and sustained the intracellular level of RA. Furthermore, the different abilities of RA and AscPNa to induce mesenchymal-epithelial transition, pluripotency, and neuronal differentiation explain their complex contribution to reprogramming when used individually or in combination. Therefore, the current studies identified a positive feedback between RA and AscPNa, or possibility between vitamin A and C, and further explored their contributions to reprogramming.

Summary: Normally, opioids function in a receptor-dependent manner. They bind to opioid receptors, activate or inhibit receptor activation, and subsequently modulate downstream signal transduction. However, the complex functions of opioids and the low expression of opioid receptors and their endogenous peptide agonists in neural stem cells (NSCs) suggest that some opioids may also modulate NSCs via a receptor-independent pathway. In the current study, two opioids, morphine and naloxone, are demonstrated to facilitate NSC proliferation via a receptor-independent and ten-eleven translocation methylcytosine dioxygenase 1 (TET1)-dependent pathway. Morphine and naloxone penetrate cell membrane, bind to TET1 protein via three key residues (1,880–1,882), and subsequently result in facilitated proliferation of NSCs. In addition, the two opioids also inhibit the DNA demethylation ability of TET1. In summary, the current results connect opioids and DNA demethylation directly at least in NSCs and extend our understanding on both opioids and NSCs. : Liang et al. demonstrate a receptor-independent and Tet1-dependent pathway used by opioids naloxone and morphine to facilitate the proliferation of NSCs. The binding of naloxone and morphine to TET1 suggests that DNA methylation should be considered when studying the tolerance- and addiction-induced opioids. Keywords: neural stem cells, naloxone, morphine, Tet1, receptor-independent, proliferation

As a critical process during embryonic development, cancer progression and cell fate conversions, epithelial-mesenchymal transition (EMT) has been extensively studied over the last several decades. To further understand the nature of EMT, we performed meta-analysis of multiple microarray datasets to identify the related generic signature. In this study, 24 human and 17 mouse microarray datasets were integrated to identify conserved gene expression changes in different types of EMT. Our integrative analysis revealed that there is low agreement among the list of the identified signature genes and three other lists in previous studies. Since removing the datasets with weakly-induced EMT from the analysis did not significantly improve the overlapping in the signature-gene lists, we hypothesized the existence of different types of EMT. This hypothesis was further supported by the grouping of 74 human EMT-induction samples into five distinct clusters, and the identification of distinct pathways in these different clusters of EMT samples. The five clusters of EMT-induction samples also improves the understanding of the characteristics of different EMT types. Therefore, we concluded the existence of different types of EMT was the possible reason for its complex role in multiple biological processes.

Background: Currently, direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds. Delivery of transcriptional factors may affect genome stability, while small-molecule compounds may require more evaluations when applied in vivo. Thus, a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation. Results: Here, we report that a defined medium (5C) consisting of basic fibroblast growth factor (bFGF), N2 supplement, leukemia inhibitory factor, vitamin C (Vc), and β-mercaptoethanol (βMe) induces the direct conversion of somatic cells to cells with neuronal characteristics. Application of 5C medium converted mouse embryonic fibroblasts (MEFs) into TuJ+ neuronal-like cells, which were capable of survival after being transplanted into the mouse brain. The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury, possibly by inducing similar conversions, when infused into the mouse brain in vivo. Crucially, 5C medium could also induce neuronal characteristics in several human cell types. Conclusions: In summary, this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.

Background: Pain management has been considered as significant contributor to broad quality-of-life improvement for cancer patients. Modulating serum cholesterol levels affects analgesia abilities of opioids, important pain killer for cancer patients, in mice system. Thus the correlation between opioids usages and cholesterol levels were investigated in human patients with lung cancer. Methods: Medical records of 282 patients were selected with following criteria, 1) signed inform consent, 2) full medical records on total serum cholesterol levels and opioid administration, 3) opioid-naïve, 4) not received/receiving cancer-related or cholesterol lowering treatment, 5) pain level at level 5-8. The patients were divided into different groups basing on their gender and cholesterol levels. Since different opioids, morphine, oxycodone, and fentanyl, were all administrated at fixed low dose initially and increased gradually only if pain was not controlled, the percentages of patients in each group who did not respond to the initial doses of opioids and required higher doses for pain management were determined and compared. Results: Patients with relative low cholesterol levels have larger percentage (11 out of 28 in female and 31 out of 71 in male) to not respond to the initial dose of opioids than those with high cholesterol levels (0 out of 258 in female and 8 out of 74 in male). Similar differences were obtained when patients with different opioids were analyzed separately. After converting the doses of different opioids to equivalent doses of oxycodone, significant correlation between opioid usages and cholesterol levels was also observed. Conclusions: Therefore, more attention should be taken to those cancer patients with low cholesterol levels because they may require higher doses of opioids as pain killer. [ABSTRACT FROM AUTHOR]

A high proliferation rate has been observed to facilitate somatic cell reprogramming, but the pathways that connect proliferation and reprogramming have not been reported. DNA methyltransferase 1 (DNMT1) methylates hemimethylated CpG sites produced during S phase and maintains stable inheritance of DNA methylation. Impairing this process results in passive DNA demethylation. In this study, we show that the cell proliferation rate positively correlated with the expression of Dnmt1 in G1 phase. In addition, as determined by wholegenome bisulfate sequencing and high-performance liquid chromatography, global DNA methylation of mouse embryonic fibroblasts was significantly higher in G1 phase than in G2/M phase. Thus, we suspected that high cellular proliferation requires more Dnmt1 expression in G1 phase to prevent passive DNA demethylation. The methylation differences of individual CpG sites betweenG1 andG2/M phase were related to the methylation status and the positions of their surrounding CpG sites. In addition, larger methylation differences were observed on the promoters of pluripotency-related genes; for example, Oct4, Nanog, Sox2, Esrrb, Cdh1, and Epcam. When such methylation differences or passive DNA demethylation accumulated with Dnmt1 suppression and proliferation acceleration, DNA methylation on pluripotency-related genes was decreased, and their expression was up-regulated, which subsequently promoted pluripotency and mesenchymal-epithelial transition, a necessary step for reprogramming. We infer that high cellular proliferation rates promote generation of induced pluripotent stem cells at least partially by inducing passive DNA demethylation and up-regulating pluripotency-related genes. Therefore, these results uncover a connection between cell reprogramming and DNA methylation. [ABSTRACT FROM AUTHOR]