Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells.

Post-treatment With Qing-Ying-Tang, a Compound Chinese Medicine Relives Lipopolysaccharide-Induced Cerebral Microcirculation Disturbance in Mice.

Objective: Lipopolysaccharide (LPS) causes microvascular dysfunction, which is a key episode in the pathogenesis of endotoxemia. This work aimed to investigate the effect of Qing-Ying-Tang (QYT), a compound Chinese medicine in cerebral microcirculation disturbance and brain damage induced by LPS. Methods: Male C57/BL6 mice were continuously transfused with LPS (7.5 mg/kg/h) through the left femoral vein for 2 h. QYT (14.3 g/kg) was given orally 2 h after LPS administration. The dynamics of cerebral microcirculation were evaluated by intravital microscopy. Brain tissue edema was assessed by brain water content and Evans Blue leakage. Cytokines in plasma and brain were evaluated by flow cytometry. Confocal microscopy and Western blot were applied to detect the expression of junction and adhesion proteins, and signaling proteins concerned in mouse brain tissue. Results: Post-treatment with QYT significantly ameliorated LPS-induced leukocyte adhesion to microvascular wall and albumin leakage from cerebral venules and brain tissue edema, attenuated the increase of MCP-1, MIP-1α, IL-1α, IL-6, and VCAM-1 in brain tissue and the activation of NF-κB and expression of MMP-9 in brain. QYT ameliorated the downregulation of claudin-5, occludin, JAM-1, ZO-1, collagen IV as well as the expression and phosphorylation of VE-cadherin in mouse brain. Conclusions: This study demonstrated that QYT protected cerebral microvascular barrier from disruption after LPS by acting on the transcellular pathway mediated by caveolae and paracellular pathway mediated by junction proteins. This result suggests QYT as a potential strategy to deal with endotoxemia.

Hilbert-Huang Transformation Based Analyses of FP1, FP2, and Fz Electroencephalogram Signals in Alcoholism.

Chronic alcoholism may damage the central nervous system, causing imbalance in the excitation-inhibition homeostasis in the cortex, which may lead to hyper-arousal of the central nervous system, and impairments in cognitive function. In this paper, we use the Hilbert-Huang transformation (HHT) method to analyze the electroencephalogram (EEG) signals from control and alcoholic observers who watched two different pictures. We examined the intrinsic mode function (IMF) based energy distribution features of FP1, FP2, and Fz EEG signals in the time and frequency domains for alcoholics. The HHT-based characteristics of the IMFs, the instantaneous frequencies, and the time-frequency-energy distributions of the IMFs of the clinical FP1, FP2, and Fz EEG signals recorded from normal and alcoholic observers who watched two different pictures were analyzed. We observed that the number of peak amplitudes of the alcoholic subjects is larger than that of the control. In addition, the Pearson correlation coefficients of the IMFs, and the energy-IMF distributions of the clinical FP1, FP2, and Fz EEG signals recorded from normal and alcoholic observers were analyzed. The analysis results show that the energy ratios of IMF4, IMF5, and IMF7 waves of the normal observers to the refereed total energy were larger than 10 %, respectively. In addition, the energy ratios of IMF3, IMF4, and IMF5 waves of the alcoholic observers to the refereed total energy were larger than 10 %. The FP1 and FP2 waves of the normal observers, the FP1 and FP2 waves of the alcoholic observers, and the FP1 and Fz waves of the alcoholic observers demonstrated extremely high correlations. On the other hand, the FP1 waves of the normal and alcoholic observers, the FP1 wave of the normal observer and the FP2 wave of the alcoholic observer, the FP1 wave of the normal observer and the Fz wave of the alcoholic observer, the FP2 waves of the normal and alcoholic FP2 observers, and the FP2 wave of the normal observer and the Fz wave of the alcoholic observer demonstrated extremely low correlations. The IMF4 of the FP1 and FP2 signals of the normal observer, and the IMF5 of the FP1 and FP2 signals of the alcoholic observer were correlated. The IMF4 of the FP1 signal of the normal observer and that of the FP2 signal of the alcoholic observer as well as the IMF5 of the FP1 signal of the normal observer and that of the FP2 signal of the alcoholic observer exhibited extremely low correlations. In this manner, our experiment leads to a better understanding of the HHT-based IMFs features of FP1, FP2, and Fz EEG signals in alcoholism. The analysis results show that the energy ratios of the wave of an alcoholic observer to its refereed total energy for IMF4, and IMF5 in the δ band for FP1, FP2, and Fz channels were larger than those of the respective waves of the normal observer. The alcoholic EEG signals constitute more than 1 % of the total energy in the δ wave, and the reaction times were 0_4, 4_8, 8_12, and 12_16 s. For normal EEG signals, more than 1 % of the total energy is distributed in the δ wave, with a reaction time 0 to 4 s. We observed that the alcoholic subject reaction times were slower than those of the normal subjects, and the alcoholic subjects could have experienced a cognitive error. This phenomenon is due to the intoxicated central nervous systems of the alcoholic subjects.

Tunable stress and controlled thickness modification in graphene by annealing.

Graphene has many unique properties which make it an attractive material for fundamental study as well as for potential applications. In this paper, we report the first experimental study of process-induced defects and stress in graphene using Raman spectroscopy and imaging. While defects lead to the observation of defect-related Raman bands, stress causes shift in phonon frequency. A compressive stress (as high as 2.1 GPa) was induced in graphene by depositing a 5 nm SiO(2) followed by annealing, whereas a tensile stress ( approximately 0.7 GPa) was obtained by depositing a thin silicon capping layer. In the former case, both the magnitude of the compressive stress and number of graphene layers can be controlled or modified by the annealing temperature. As both the stress and thickness affect the physical properties of graphene, this study may open up the possibility of utilizing thickness and stress engineering to improve the performance of graphene-based devices. Local heating techniques may be used to either induce the stress or reduce the thickness selectively.