Transcriptomic profiling across human serotonin neuron differentiation via the FEV reporter system.

BACKGROUND: The detailed transcriptomic profiles during human serotonin neuron (SN) differentiation remain elusive. The establishment of a reporter system based on SN terminal selector holds promise to produce highly-purified cells with an early serotonergic fate and help elucidate the molecular events during human SN development process. METHODS: A fifth Ewing variant (FEV)-EGFP reporter system was established by CRISPR/Cas9 technology to indicate SN since postmitotic stage. FACS was performed to purify SN from the heterogeneous cell populations. RNA-sequencing analysis was performed for cells at four key stages of differentiation (pluripotent stem cells, serotonergic neural progenitors, purified postmitotic SN and purifed mature SN) to explore the transcriptomic dynamics during SN differentiation. RESULTS: We found that human serotonergic fate specification may commence as early as day 21 of differentiation from human pluripotent stem cells. Furthermore, the transcriptional factors ZIC1, HOXA2 and MSX2 were identified as the hub genes responsible for orchestrating serotonergic fate determination. CONCLUSIONS: For the first time, we exposed the developmental transcriptomic profiles of human SN via FEV reporter system, which will further our understanding for the development process of human SN.

Corticosterone Impairs Hippocampal Neurogenesis and Behaviors through p21-Mediated ROS Accumulation.

Stress is known to induce a reduction in adult hippocampal neurogenesis (AHN) and anxiety-like behaviors. Glucocorticoids (GCs) are secreted in response to stress, and the hippocampus possesses the greatest levels of GC receptors, highlighting the potential of GCs in mediating stress-induced hippocampal alterations and behavior deficits. Herein, RNA-sequencing (RNA-seq) analysis of the hippocampus following corticosterone (CORT) exposure revealed the central regulatory role of the p21 (Cdkna1a) gene, which exhibited interactions with oxidative stress-related differentially expressed genes (DEGs), suggesting a potential link between p21 and oxidative stress-related pathways. Remarkably, p21-overexpression in the hippocampal dentate gyrus partially recapitulated CORT-induced phenotypes, including reactive oxygen species (ROS) accumulation, diminished AHN, dendritic atrophy, and the onset of anxiety-like behaviors. Significantly, inhibiting ROS exhibited a partial rescue of anxiety-like behaviors and hippocampal alterations induced by p21-overexpression, as well as those induced by CORT, underscoring the therapeutic potential of targeting ROS or p21 in the hippocampus as a promising avenue for mitigating anxiety disorders provoked by chronic stress.

A Novel Dextran-Based Dual Drug Conjugate Targeted Tumors with High Biodistribution Ratio of Tumors to Normal Tissues.

Purpose: Most chemotherapeutic agents possess poor water solubility and show more significant accumulations in normal tissues than in tumor tissues, resulting in serious side effects. To this end, a novel dextran-based dual drug delivery system with high biodistribution ratio of tumors to normal tissues was developed. Methods: A bi-functionalized dextran was developed, and several negatively charged dextran-based dual conjugates containing two different types of drugs, docetaxel and docosahexaenoic acid (DTX and DHA, respectively) were synthesized. The structures of these conjugates were characterized using nuclear magnetic resonance and liquid chromatography/mass spectrometry (1H-NMR and LC/MS, respectively) analysis. Cell growth inhibition, apoptosis, cell cycle distribution, and cellular uptake were measured in vitro. Drug biodistribution and pharmacokinetics were investigated in mice bearing 4T1 tumors using LC/MS analysis. Drug biodistribution was also explored by in vivo imaging. The effects of these conjugates on tumor growth were evaluated in three mice models. Results: The dextran-docosahexaenoic acid (DHA)- docetaxel (DTX) conjugates caused a significant enhancement of DTX water solubility and improvement in pharmacokinetic characteristics. The optimized dextran-DHA-DTX conjugate A treatment produced a 2.1- to 15.5-fold increase in intra-tumoral DTX amounts for up to 96 h compared to parent DTX treatment. Meanwhile, the concentrations of DTX released from conjugate A in normal tissues were much lower than those of the parent DTX. This study demonstrated that DHA could lead to an improvement in the efficacy of the conjugates and that the conjugate with the shortest linker displayed more activity than conjugates with longer linkers. Moreover, conjugate A completely eradicated all MCF-7 xenograft tumors without causing any obvious side effects and totally outperformed both the conventional DTX formulation and Abraxane in mice. Conclusion: These dextran-based dual drug conjugates may represent an innovative tumor targeting drug delivery system that can selectively deliver anticancer agents to tumors.

The enhanced antitumor activity of the polymeric conjugate covalently coupled with docetaxel and docosahexaenoic acid.

Docetaxel (DTX) has been widely used for the treatment of many types of cancer. However, DTX is poorly water-soluble and commercial DTX is formulated in non-ionic surfactant polysorbate 80 and ethanol, thereby leading to hypersensitivity and serious side effects. Herein, a polymer dual drug conjugate was synthesized by coupling DTX and docosahexaenoic acid (DHA) with bifunctionalized dextran. The polysaccharide conjugate dextran-DHA-DTX possessed high water solubility and was self-assembled into nanoparticles with a diameter of 98.0 ± 6.4 nm. Pharmacokinetic and biodistribution studies showed that the dextran-DHA-DTX dual drug conjugate not only had significantly prolonged blood circulation but was also selectively accumulated in the tumor with reduced drug distribution in normal tissues. The conjugate exhibited a superior therapeutic effect in both xenograft nude mice models without causing any systemic side effects. Notably, the conjugate nearly eliminated all xenograft tumors in nude mice bearing breast cancer cells MCF-7. This study revealed that the dextran-based dual drug delivery system may provide an effective strategy to selectively deliver DTX to tumor sites.

Cisplatin enhances protein O­GlcNAcylation by altering the activity of OGT, OGA and AMPK in human non­small cell lung cancer cells.

O­GlcNAcylation is a dynamic and reversible post­translational modification of proteins that is modulated by O­GlcNAc transferase (OGT) and O­GlcNAcase (OGA). Alterations in the protein expression of O­linked ß­N­acetylglucosamine (O­GlcNAc) can be induced by multiple factors. However, little is known of the effects of chemotherapeutic agents on O­GlcNAcylation and the relevant molecular mechanisms in cancer cells. In the present study, to investigate whether cisplatin alters protein O­GlcNAcylation and to explore whether protein O­GlcNAc modification affects the antitumor activity of cisplatin, experiments were performed in vitro and in vivo. The results indicated that cisplatin treatment resulted in an enhancement of global protein O­GlcNAc levels in the H1299, Hep G2 and MCF­7 cells in vitro and in vivo. Cisplatin upregulated the protein and mRNA expression levels of OGT and OGA in H1299 cells. Moreover, cisplatin induced the significant enhancement of the enzymatic activity of OGT in H1299 cells. On the contrary, the activation of OGA decreased in response to cisplatin exposure in H1299 cells. Cisplatin inhibited the activity of AMP­activated protein kinase (AMPK) by decreasing the AMP/ATP ratio. The present study also revealed that the decreased AMPK activation inhibited glutamine­fructose­6­phosphate aminotransferase (isomerizing) 1 (GFAT1) phosphorylation and subsequently promoted the activity of GFAT1. Cisplatin­induced GFAT1 activation elevated the production of the donor substrate, uridine 5­diphospho­N­acetylglucosamine (UDP­GlcNAc). However, alterations in the O­GlcNAc levels by the inhibition of OGT and OGA did not affect the sensitivity of lung cancer cells to cisplatin. On the whole, the present study demonstrates that cisplatin enhances protein O­GlcNAcylation by altering the activity of OGT, OGA and AMPK in vitro and in vivo.