Real-Time Monitoring of Tyrosine Hydroxylase Activity with a Ratiometric Fluorescent Probe.

Parkinson's disease (PD) represents the second most widespread neurodegenerative disease, and early monitoring and diagnosis are urgent at present. Tyrosine hydroxylase (TH) is a key enzyme for producing dopamine, the levels of which can serve as an indicator for assessing the severity and progression of PD. This renders the specific detection and visualization of TH a strategically vital way to meet the above demands. However, a fluorescent probe for TH monitoring is still missing. Herein, three rationally designed wash-free ratiometric fluorescent probes were proposed. Among them, TH-1 exhibited ideal photophysical properties and specific dual-channel bioimaging of TH activity in SH-SY5Y nerve cells. Moreover, the probe allowed for in vivo imaging of TH activity in zebrafish brain and living striatal slices of mice. Overall, the ratiometric fluorescent probe TH-1 could serve as a potential tool for real-time monitoring of PD in complex biosystems.

Evaluation of the topographical influence on the cellular behavior of human umbilical vein endothelial cells.

Adhesion and proliferation of vascular endothelial cells are important parameters in the endothelialization of biomedical devices for vascular applications. Endothelialization is a complex process affected by endothelial cells and their interaction with the extracellular microenvironment. Although numerous approaches are taken to study the influence of the external environment, a systematic investigation of the impact of an engineered microenvironment on endothelial cell processes is needed. This study aims to investigate the influence of topography, initial cell seeding density, and collagen coating on human umbilical vein endothelial cells (HUVECs). Utilizing the MultiARChitecture (MARC) chamber, the effects of various topographies on HUVECs are identified, and those with more prominent effects were further evaluated individually using the MARC plate. Endothelial cell marker expression and monocyte adhesion assay are examined on the HUVEC monolayer. HUVECs on 1.8 µm convex and concave microlens topographies demonstrate the lowest cell adhesion and proliferation, regardless of initial cell seeding density and collagen I coating, and the HUVEC monolayer on the microlens shows the lowest monocyte adhesion. This property of lens topographies would potentially be a useful parameter in designing vascular biomedical devices. The MARC chamber and MARC plate show a great potential for faster and easy pattern identification for various cellular processes.

Microlens topography combined with vascular endothelial growth factor induces endothelial differentiation of human mesenchymal stem cells into vasculogenic progenitors.

Cell therapy for vascular damage has been showing promises as alternative therapy for endothelial dysfunctions since the discovery of the endothelial progenitor cells (EPCs). However, isolated EPCs from peripheral blood yield low cell amounts and alternative cell source must be explored. The aim of this study was to investigate the influence of topography on the endothelial differentiation of an alternative cell source - human mesenchymal stem cells (hMSCs) from bone marrow. Utilizing the MultiARChitecture (MARC) chip, a systematic screening of variety of patterned surfaces and different medium compositions was performed. While topographical patterns alone induce endothelial differentiation, a synergistic enhancement was observed when topography was combined with a medium enriched with vascular endothelial growth factor (VEGF). The 1.8 µm diameter convex microlens pattern in combination with the VEGF enriched medium was shown to be the most efficient on the endothelial differentiation, yielding up to 10% of CD34+CD133+KDR+ marker expressing differentiated hMSCs as analyzed by flow cytometry. The quantified tube-like structures in the Matrigel assay in vitro indicated a vasculogenic potential of these endothelial progenitor-like differentiated hMSCs that was investigated further in a Matrigel plug assay in vivo in a rat for seven days. Explanted Matrigel plugs were processed with hematoxylin-eosin (H&E) and anti-Ulex Europaeus agglutinin (UEA-1) staining to visualize the capillaries and to identify the presence of human cells. The hMSCs cultured on the 1.8 µm diameter convex microlens in a medium enriched with VEGF, implanted in a Matrigel plug in a rat, showed the highest capillary density, the highest UEA-1+ capillary density, as well as the highest UEA-1+ cell survival density that were not included in the vasculogenesis. These findings indicate the active participation of the vasculogenic hMSCs in the vasculogenesis. The endothelial differentiation of hMSCs using this synergistic combination of microlens and VEGF enriched medium was also demonstrated in hMSCs from different male and female donors. The culture platform with combination of topography and biochemical cues could generate vasculogenic cell populations that may prove useful in vascular damage or other clinical applications.

H3K27 trimethylation is an early epigenetic event of p16INK4a silencing for regaining tumorigenesis in fusion reprogrammed hepatoma cells.

Stable epigenetic silencing of p16(INK4a) is a common event in hepatocellular carcinoma (HCC) cells, which is associated with abnormal cell proliferation and liberation from cell cycle arrest. Understanding the early epigenetic events in silencing p16(INK4a) expression may illuminate a prognostic strategy to block HCC development. Toward this end, we created a reprogram cell model by the fusion mouse HCC cells with mouse embryonic stem cells, in which the ES-Hepa hybrids forfeited HCC cell characteristics along with reactivation of the silenced p16(INK4a). HCC characteristics, in terms of gene expression pattern and tumorigenic potential, was restored upon induced differentiation of these reprogrammed ES-Hepa hybrids. The histone methylation pattern relative to p16(INK4a) silencing during differentiation of the ES-Hepa hybrids was analyzed. H3K27 trimethylation at the p16(INK4a) promoter region, occurring in the early onset of p16(INK4a) silencing, was followed by H3K9 dimethylation at later stages. During the induced differentiation of the ES-Hepa hybrids, H3K4 di- and trimethylations were maintained at high levels during the silencing of p16(INK4a), strongly suggesting that H3K4 methylation events did not cause the silencing of p16(INK4a). Our results suggested that the enrichment of H3K27 trimethylation, independent of H3K9 dimethylation, trimethylation, and DNA methylation, was an early event in the silencing of p16(INK4a) during the tumor development. This unique chromatin pattern may be a heritable marker of epigenetic regulation for p16(INK4a) silencing during the developmental process of hepatocellular carcinogenesis.

[Small interfering RNA-mediated nuclear factor-kappaB P65 suppression induces apoptosis of hepatic carcinoma SMMC-7721 cells].

OBJECTIVE: To study the mechanism of hepatic carcinoma cell apoptosis induced by small interfering RNA (siRNA)-mediated nuclear factor-kappaB (NF-kappaB) P65 silencing. METHODS: Hepatic carcinoma SMMC-7721 cells were exposed to liposome-mediated transfection with NF-kappaB P65 siRNA synthesized by in vitro transcription, and the cells with empty liposome transfection and those without particular treatment served as the control groups. The expression of NF-kappaB P65 in the cells was detected by Western blotting, the cell viability examined by MTT assay, and the cell apoptosis assessed by flow cytometry. Immunohistochemistry was used to examine the expressions of Bcl-2 and Bax. RESULTS: siRNA transfection significantly inhibited the expression of NF-kappaB P65 in SMMC-7721cells, with inhibition rates of 64.74% compared with the untreated cells and of 34.52% compared with the liposome-treated cells. The siRNA-treated SMMC-7721 cells also exhibited significant decrease in cell proliferation by 33.39% and 27.23% in comparison with the untreated and liposome-treated cells, respectively. NF-kappaB P65 siRNA induced obvious cell apoptosis with down-regulated Bcl-2 and up-regulated Bax expressions. CONCLUSION: NF-kappaB p65 siRNA can induce SMMC-7721 cell apoptosis via the Bcl-2/Bax pathway.