Generation of a stably transfected mouse embryonic stem cell line for inducible differentiation to excitatory neurons.

In vitro differentiation of stem cells into various cell lineages is valuable in developmental studies and an important source of cells for modelling physiology and pathology, particularly for complex tissues such as the brain. Conventional protocols for in vitro neuronal differentiation often suffer from complicated procedures, high variability and low reproducibility. Over the last decade, the identification of cell fate-determining transcription factors has provided new tools for cellular studies in neuroscience and enabled rapid differentiation driven by ectopic transcription factor expression. As a proneural transcription factor, Neurogenin 2 (Ngn2) expression alone is sufficient to trigger rapid and robust neurogenesis from pluripotent cells. Here, we established a stable cell line, by piggyBac (PB) transposition, that conditionally expresses Ngn2 for generation of excitatory neurons from mouse embryonic stem cells (ESCs) using an all-in-one PB construct. Our results indicate that Ngn2-induced excitatory neurons have mature and functional characteristics consistent with previous studies using conventional differentiation methods. This approach provides an all-in-one PB construct for rapid and high copy number gene delivery of dox-inducible transcription factors to induce differentiation. This approach is a valuable in vitro cell model for disease modeling, drug screening and cell therapy.

Constructing a competitive endogenous RNA network of EndMT-related atherosclerosis through weighted gene co-expression network analysis.

Atherosclerosis is a chronic inflammatory disease characterized by endothelial dysfunction and plaque formation. Under pro-inflammatory conditions, endothelial cells can undergo endothelial-to-mesenchymal transition (EndMT), contributing to atherosclerosis development. However, the specific regulatory mechanisms by which EndMT contributes to atherosclerosis remain unclear and require further investigation. Dan-Shen-Yin (DSY), a traditional Chinese herbal formula, is commonly used for cardiovascular diseases, but its molecular mechanisms remain elusive. Emerging evidence indicates that competing endogenous RNA (ceRNA) networks play critical roles in atherosclerosis pathogenesis. In this study, we constructed an EndMT-associated ceRNA network during atherosclerosis progression by integrating gene expression profiles from the Gene Expression Omnibus (GEO) database and weighted gene co-expression network analysis. Functional enrichment analysis revealed this EndMT-related ceRNA network is predominantly involved in inflammatory responses. ROC curve analysis showed the identified hub genes can effectively distinguish between normal vasculature and atherosclerotic lesions. Furthermore, Kaplan-Meier analysis demonstrated that high expression of IL1B significantly predicts ischemic events in atherosclerosis. Molecular docking revealed most DSY bioactive components can bind key EndMT-related lncRNAs, including AC003092.1, MIR181A1HG, MIR155HG, WEE2-AS1, and MIR137HG, suggesting DSY may mitigate EndMT in atherosclerosis by modulating the ceRNA network.

Network pharmacology and experimental analysis to reveal the mechanism of Dan-Shen-Yin against endothelial to mesenchymal transition in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease characterized by the formation of plaque and endothelial dysfunction. Under pro-inflammatory conditions, endothelial cells adopt a mesenchymal phenotype by a process called endothelial-to-mesenchymal transition (EndMT) which plays an important role in the pathogenesis of atherosclerosis. Dan-Shen-Yin (DSY) is a well-known traditional Chinese medicine used in the treatment of cardiovascular disease. However, the molecular mechanism whereby DSY mitigates atherosclerosis remains unknown. Therefore, we employed a network pharmacology-based strategy in this study to determine the therapeutic targets of DSY, and in vitro experiments to understand the molecular pharmacology mechanism. The targets of the active ingredients of DSY related to EndMT and atherosclerosis were obtained and used to construct a protein-protein interaction (PPI) network followed by network topology and functional enrichment analysis. Network pharmacology analysis revealed that the PI3K/AKT pathway was the principal signaling pathway of DSY against EndMT in atherosclerosis. Molecular docking simulations indicated strong binding capabilities of DSY's bioactive ingredients toward PI3K/AKT pathway molecules. Experimentally, DSY could efficiently modify expression of signature EndMT genes and decrease expression of PI3K/AKT pathway signals including integrin αV, integrin ß1, PI3K, and AKT1 in TGF-ß2-treated HUVECs. LASP1, which is upstream of the PI3K/AKT pathway, had strong binding affinity to the majority of DSY's bioactive ingredients, was induced by EndMT-promoting stimuli involving IL-1ß, TGF-ß2, and hypoxia, and was downregulated by DSY. Knock-down of LASP1 attenuated the expression of integrin αV, integrin ß1, PI3K, AKT1 and EndMT-related genes induced by TGF-ß2, and minimized the effect of DSY. Thus, our study showed that DSY potentially exerted anti-EndMT activity through the LASP1/PI3K/AKT pathway, providing a possible new therapeutic intervention for atherosclerosis.

Efficient Generation of Stable Cell Lines with Inducible Neuronal Transgene Expression Using the piggyBac Transposon System.

The piggyBac transposon system has been adapted to be a highly efficient genome engineering tool for transgenesis of eukaryotic cells and organisms. As with other methods of transgenesis, incorporation of an inducible promoter, such as a tetracycline-responsive element, enables inducible transgene expression. Here, we describe an efficient method of using the piggyBac system to create stably transfected mammalian cell lines, including inducible transgene expression. Gibson assembly is used to construct the required vectors as it enables multiple DNA fragments to be seamlessly assembled in a single isothermal reaction. We demonstrate an application of this approach to generate a stably transfected pluripotent stem cell line that can be induced to express a transcription factor transgene and rapidly differentiate into neurons in a single step.

Targeting the AAVS1 Site by CRISPR/Cas9 with an Inducible Transgene Cassette for the Neuronal Differentiation of Human Pluripotent Stem Cells.

CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.

Effects Of Minocycline Combined With Tinidazole For Treatment Of Chronic Periodontitis.

Purpose: To investigate the therapeutic effects of minocycline combined with tinidazole in the treatment of chronic periodontitis (CP). Methods: Seventy-three CP patients treated May 2018­December 2019 at Yuyao People's Hospital (Yuyao, China) were enrolled in this study: 34 were treated with minocycline alone (control group; CG) and 39 were treated with a combination of minocycline and tinidazole (observation group; OG). Both groups were treated continuously for four weeks and plaque index (PLI), bleeding index (BI), periodontal pocket depth (PD), periodontal attachment level (PAL) and alveolar bone height were compared before and after treatment. Pain was evaluated using the visual analogue scale (VAS). Levels of TNF-α and IL-6 before and after treatment were determined using an enzyme-linked immunosorbent assay. Adverse reactions were compared. Results: In each group, PLI, BI, PD, PAL and alveolar bone height were lower after treatment (P<0.05), and those in OG were lower than those in CG (P<0.05). TNF-α and IL-6 levels in both groups were lower after treatment (P<0.05), and the levels in serum of the OG were lower than those of the CG (P<0.05). After treatment, the VAS in OG was lower than that of CG (P<0.05). There was no significant difference in adverse reactions between groups (P>0.05). Conclusion: Minocycline combined with tinidazole was more effective in treating CP than minocycline alone. This drug combination improved the periodontal indexes and inflammatory reaction of CP and relieved their pain. No significant difference in adverse reactions was seen.

Forward Programming of Pluripotent Stem Cells to Neurons.

Pluripotent stem cells (PSCs) are powerful tools for studying developmental biology and neuronal diseases. Conventional differentiation protocols require several intermediate states and different culture conditions, inefficiently generating mixed subtypes of neuronal cells with immature characteristics. Direct programming of PSCs by forced expression of neuronal transcription factors has shown rapid cell fate determination with high purity as it can bypass sequential developmental steps that traditional culture requires. In this review, we focus on neuronal differentiation from PSCs to specific subtypes by various transcription factors. Furthermore, the potential applications and limitations of this novel technology are discussed.