Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Background

Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H₂O₂).

Results

Using geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H₂O₂. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.

Conclusion

Taken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

     

秦岭辛家山林区锐齿栎外生菌根真菌多样性

以秦岭辛家山林区锐齿栎为研究对象,通过野外调查结合形态学和分子生物学方法,观察和鉴定与其共生的外生菌根真菌。结果确定了3种子囊菌和48种担子菌,分属于10科14属,其中毛革菌属Tomentella是优势类群,丝伞盖属1 Inocybe 1、Russula persicina、毛革菌属1 Tomentella 1、毛革菌属2 Tomentella 2、块菌属Tuber、绒盖牛肝菌属Xerocomus是常见种,其余都为稀有种。两样地锐齿栎外生菌根真菌除丰富度指数外,Simpson指数、Shannon指数以及Pielou均匀度指数差异不显著;Jaccard相似性指数和Sorenson相似性指数分别为0.1154和0.2069。     

Pretreatments of Broussonetia papyrifera: in vitro assessment on gas and methane production,fermentation characteristic,and methanogenic archaea profile

ObjectiveThe present study was conducted to examine the gas production, fermentation characteristics, nutrient degradation, and methanogenic community composition of a rumen fluid culture with Broussonetia papyrifera (B. papyrifera) subjected to ensiling or steam explosion (SE) pretreatment.MethodsFresh B. papyrifera was collected and pretreated by ensiling or SE, which was then fermented with ruminal fluids as ensiled B. papyrifera group, steam-exploded B. papyrifera group, and untreated B. papyrifera group. The gas and methane production, fermentation characteristics, nutrient degradation, and methanogenic community were determined during the fermentation.ResultsCumulative methane production was significantly improved with SE pretreatment compared with ensiled or untreated biomass accompanied with more volatile fatty acids production. After 72 h incubation, SE and ensiling pretreatments decreased the acid detergent fiber contents by 39.4% and 22.9%, and neutral detergent fiber contents by 10.6% and 47.2%, respectively. Changes of methanogenic diversity and abundance of methanogenic archaea corresponded to the variations in fermentation pattern and methane production.ConclusionCompared with ensiling pretreatment, SE can be a promising technique for the efficient utilization of B. papyrifera, which would contribute to sustainable livestock production systems.     

Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners

It has been reported that it is difficult to express cationic antibacterial peptides in engineered bacteria because such peptides are highly toxic to the host bacteria cells and sensitive to intracellular proteases. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells, which may possibly be used as an antimicrobial agent. Here we tried to express ABP-CM4 in Escherichia coli cells using either the GST fusion system or the intein-mediated fusion expression system. In order to investigate the possible use of these two fusion partners in cationic small peptide expression and purification, a mutant ABP-CMt, which is a highly positively charged peptide with +9 charges at neutral pH, was designed. In the present study, we have shown that both ABP-CM4 and ABP-CMt peptides can be expressed and purified by the intein-mediated expression system but not by the GST fusion expression system. Thus the intein-mediated peptide expression and purification system potentially could be employed for the production of recombinant protease-sensitive and cytotoxic peptides.     

Effect of Nrf2 signaling pathway on the improvement of intestinal epithelial barrier dysfunction by hyperbaric oxygen treatment after spinal cord injury

Disruption of the intestinal epithelial barrier following spinal cord injury (SCI) seriously affect long-term quality of life. Oxidative stress-induced epithelial cells’ injury contributes to the epithelial barrier dysfunction. Hyperbaric oxygen (HBO) treatment has been proved to alleviate SCI. However, it is unclear whether or not HBO treatment affects intestinal barrier function following SCI. In this study, our purpose was to explore the impact of HBO treatment on intestinal epithelial barrier function and underlying mechanisms following SCI. An SCI model was established in rats, and the rats received HBO treatment. Intestinal injury, mucosal permeability, intercellular junction proteins, and oxidative stress indicators were evaluated in our study. We found that HBO treatment significantly alleviated intestinal histological damage, reduced mucosal permeability, and markedly prevented bacterial translocation. Furthermore, HBO treatment significantly increased the expression of Claudin-1 and E-cadherin, inhibited intestinal tissue oxidative stress as demonstrated by upregulation of superoxide dismutase and glutathione, and HBO downregulated malondialdehyde. Mechanically, we demonstrated that HBO treatment ameliorated intestinal oxidative stress possibly through upregulating nuclear factor E2-related factor 2 (Nrf2) and its downstream targets, Heme oxygenase-1(HO-1), NADH-quinone oxidoreductase-1(NQO-1), and glutamate cysteine ligase catalytic subunit (GCLC). These results suggested that HBO treatment triggered antioxidative effects against intestinal epithelial barrier dysfunction by promoting Nrf2 signaling pathway after SCI.