Long-term in vivo gene expression in mouse kidney using ϕC31 integrase and electroporation

Background: Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes.

Purpose: The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using ?C31 integrase.

Methods: Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5?µg of pORF-luc/attB as a donor plasmid and 20?µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression.

Results: Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, ?C31 integrase-transfected cells had less genomic DNA damage caused by integrase expression.

Discussion and conclusion: These results demonstrated that the ?C31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.     

Effects of Oxytocin on Attention to Emotional Faces in Healthy Volunteers and Highly Socially Anxious Males

Background:

Evidence suggests that individuals with social anxiety demonstrate vigilance to social threat, whilst the peptide hormone oxytocin is widely accepted as supporting affiliative behaviour in humans.

Methods:

This study investigated whether oxytocin can affect attentional bias in social anxiety. In a double-blind, randomized, placebo-controlled, within-group study design, 26 healthy and 16 highly socially anxious (HSA) male volunteers (within the HSA group, 10 were diagnosed with generalized social anxiety disorder) were administered 24 IU of oxytocin or placebo to investigate attentional processing in social anxiety. Attentional bias was assessed using the dot-probe paradigm with angry, fearful, happy and neutral face stimuli.

Results:

In the baseline placebo condition, the HSA group showed greater attentional bias for emotional faces than healthy individuals. Oxytocin reduced the difference between HSA and non-socially anxious individuals in attentional bias for emotional faces. Moreover, it appeared to normalize attentional bias in HSA individuals to levels seen in the healthy population in the baseline condition. The biological mechanisms by which oxytocin may be exerting these effects are discussed.

Conclusions:

These results, coupled with previous research, could indicate a potential therapeutic use of this hormone in treatment for social anxiety.     

Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

Aim:

To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target.

Methods:

α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis.

Results:

Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel.

Conclusion:

We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.     

Toxicological effects of nickel chloride on the cytokine mRNA expression and protein levels in intestinal mucosal immunity of broilers

The purpose of this study was to examine the toxicological effects of nickel chloride (NiCl₂; 300, 600, and 900 mg kg^?1 diet) on the cytokine mRNA expression and protein levels in the intestinal mucosa and cecal tonsil, and on the ileac and cecal tonsil T cells in broilers by the methods of qRT‐PCR, flow cytometry and ELISA for 42 days. Results showed that the IL‐2, IL‐6, IL‐10, IL‐17, IFN‐γ, and TNF‐α (LITAF) cytokine mRNA expression and protein levels were lower (P < 0.05 or P < 0.01) and the percentages of T‐cell subsets were also lower in the 300, 600, and 900 mg kg^?1 groups than in the control group. It was concluded that dietary NiCl₂ in excess of 300 mg kg^?1 could reduce cytokine mRNA expression and protein levels in the intestinal mucosa and cecal tonsil, and the percentages of ileac and cecal tonsil T‐cell subsets. Decreasing in cytokine mRNA expression and protein levels of intestinal mucosa and cecal tonsil induced by NiCl₂ was closely related to the reduction of T‐cell population. Thus, the abnormal expression of these cytokines impacts the intestinal mucosal immune function by the pathways of reducing of lymphocyte population and activation. Also, this study first proved that NiCl₂ at higher levels has the toxicological effects on intestinal mucosal immunity. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1309–1321, 2015.     

Differential effects of route of T‐2 toxin exposure on hepatic oxidative damage in mice

T‐2 toxin is the most toxic among mycotoxins and poses a potential health hazard for both humans and animals. At high doses, T‐2 toxin can cause shock‐like syndrome that can result in death. We evaluated the effect of time course and route of exposure on hepatic oxidative damage in mice and it is only such study so far to compare the effects of dermal and subcutaneous exposure of T‐2 toxin. Mice were exposed to 1 LD50 of T‐2 toxin either by percutaneous (5.94 mg/kg body weight) or subcutaneous (1.54 mg/kg body weight) route and sacrificed at 0, 1, 3, and 7 days postexposure. Analysis of a number of serum biochemical variables, antioxidant enzymes activity, gene and protein expression by immunoblot assay showed time and route dependent effects of T‐2 induced hepatic oxidative damage. Time dependent increase in protein carbonyl content and protein oxidation was seen in serum and liver. Results of our study may provide possible mechanism for developing medical countermeasures against T‐2 toxin. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 64–73, 2015.     

RNA干扰在白血病基因治疗中的应用前景

RNA干扰(RNAi)作为一种进化保守的转录后基因沉默机制,是指双链RNA分子(dsRNA)被切割成21～23个核苷酸的小干扰RNA(siRNA),最终使其同源的mRNA特异性降解.RNAi现广泛用于多个生物体的功能基因组学研究,以及在基因表达异常导致的多种疾病的临床前模型中进行干预治疗.大约50%的白血病伴有异常基因的表达,利用RNAi肿瘤特异性基因靶向治疗为治疗白血病提供了一条新途径.随着siRNA在活体中的稳定性和转染效率的提高、非特异性作用的减少,正迅速应用于临床中.