Rabies Prophylactic and Treatment Options: An In Vitro Study of siRNA- and Aptamer-Based Therapeutics

If the goal of eliminating dog-mediated human rabies by 2030 is to be achieved, effective mass dog vaccination needs to be complemented by effective prophylaxis for individuals exposed to rabies. Aptamers and short-interfering RNAs (siRNAs) have been successful in therapeutics, but few studies have investigated their potential as rabies therapeutics. In this study, siRNAs and aptamers—using a novel selection method—were developed and tested against rabies virus (RABV) in a post-infection (p.i.) scenario. Multiple means of delivery were tested for siRNAs, including the use of Lipofectamine and conjugation with the developed aptamers. One siRNA (N53) resulted in an 80.13% reduction in viral RNA, while aptamer UPRET 2.03 demonstrated a 61.3% reduction when used alone at 2 h p.i. At 24 h p.i., chimera UPRET 2.03-N8 (aptamer-siRNA) resulted in a 36.5% inhibition of viral replication. To our knowledge, this is the first study using siRNAs or aptamers that (1) demonstrated significant inhibition of RABV using an aptamer, (2) tested Lipofectamine RNAi-Max as a means for delivery, and (3) produced significant RABV inhibition at 24 h p.i. This study serves as a proof-of-concept to potentially use aptamers and siRNAs as rabies immunoglobulin (RIG) replacements or therapeutic options for RABV and provides strong evidence towards their further investigation.     

p21基因沉默联合表阿霉素促进乳腺癌MCF-7细胞凋亡及其机制的研究

目的研究si RNA沉默p21基因表达联合表阿霉素对MCF-7细胞增殖和凋亡的影响及相关作用机制。方法将化学合成的针对p21的si RNA序列转染至MCF-7细胞,将MCF-7细胞分为5组:空白对照组、阴性对照组、RNA抑制组、表阿霉素组、联合组(RNA沉默+表阿霉素)。采用MTT比色法、流式细胞术和分光光度法分别检测MCF-7细胞增殖、细胞凋亡、细胞周期及半胱氨酸天冬氨酸蛋白酶家族成员Caspase-9、Caspase-3和Caspase-6的活化程度。结果 p21 si RNA转染后,相对于表阿霉素单独处理组,联合组细胞死亡率明显升高(P0.01);Caspase-9、Caspase-3和Caspase-6的活化程度显著升高(P0.01)。结论 p21靶向RNA抑制联合表阿霉素处理显著促进了MCF-7细胞凋亡,p21可作为乳腺癌基因治疗的后选新靶点。     

Dietary ellagic acid improves oxidant-induced endothelial dysfunction and atherosclerosis: Role of Nrf2 activation

Background

Oxidative stress-induced vascular endothelial cell injury is a major factor in the pathogenesis of atherosclerosis. Several evidences indicate that ellagic acid (EA), a phenolic compound, contributes to cardiovascular health. This study was to investigate the effects of EA on endothelial dysfunction and atherosclerosis via antioxidant-related mechanisms.

Methods

In animal studies, wild-type (WT) C57BL/6 mice and apolipoprotein E-deficient mice (ApoE^−/−) mice were fed: a high-fat (21%) diet (HFD) or a HFD plus with EA (HFD + EA), for 14 weeks. Vascular reactivity was studied in mice aortas. The effect of EA in human umbilical vein endothelial cells (HAECs) exposed to hypochlorous acid (HOCl) was also investigated.

Results

Compared with animals on HFD alone, EA attenuated atherosclerosis in WT mice. In aortic rings from two mice models, EA significantly improved endothelium-dependent relaxation and attenuated HOCl-induced endothelial dysfunction. Besides, EA significantly improved nitric oxide synthase activity, antioxidant capacity and markers of endothelial dysfunction in plasma. Western blot analysis showed that EA increased NF-E2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1) expression in the aortas (P < 0.05). In a separate experiment, EA did not protect against HOCl-induced endothelial dysfunction in arteries obtained from Nrf2 gene knockout mice compared with WT mice. In HAECs, EA prevented HOCl-induced cellular damage and induced HO-1 protein expression, and these effects markedly abolished by the siRNA of Nrf2.

Conclusions

Our results provide further support for the protective effects of dietary EA particularly oxidant-induced endothelial dysfunction and atherosclerosis partly via Nrf2 activation.     

Graphene quantum dots: Synthesis,characterization, cell viability,genotoxicity for biomedical applications

We report the synthesis and applications of a novel N-doped graphene quantum dots (GQDs) using hydrothermal reaction between citric acid and p-aminophenol. The synthesized N-doped GQDs have been characterized physico-chemically and evaluated its antioxidant, antimicrobial, DNA binding and cleavage activities. siRNA loading studies were performed and their effects on cells were evaluated. Obtained results indicate that monodisperse solution of N-doped GQDs has been obtained with particles size ca. ～10.9 ± 1.3 nm. UV–Vis spectroscopy studies of the interactions between the N-doped GQDs and calf thymus DNA (CT-DNA) showed that the compound interact with CT-DNA via both intercalative and electrostatic binding. The DNA cleavage study showed that the N-doped GQDs cleaved DNA without any external agents. The antioxidant activity of N-doped GQDS was very active when compared to BHT. As the concentration of the compound increased, the antioxidant activity also increased. Cell viability assay demonstrated that the Ndoped GQDs showed cell viability (70%) when the concentration reached 200 μg/mL for A549 and also MDA-MB-231, 150 μg/mL for NIH-3T3 cell lines at 24 h incubation. N-doped GQDs were coated with Eudragit RS 100 and EphA2-siRNA was loaded. As a result of the studies on these formulations, it was concluded that there may be significant effects on A549 cells. The microscopy results revealed that N-doped GQDs was quickly internalized into the cell. Our novel N-doped-GQDs with siRNA are candidate for in situ tumor suppression via DNA and mRNA breakage.     

High-content analysis for mitophagy response to nanoparticles: A potential sensitive biomarker for nanosafety assessment

Mitophagy, a selective autophagy of mitochondria, clears up damaged mitochondria to maintain cell homeostasis. We performed high-content analysis (HCA) to detect the increase of PINK1, an essential protein controlling mitophagy, in hepatic cells treated with several nanoparticles (NPs). PINK1 immunofluorescence-based HCA was more sensitive than assays and detections for cell viability and mitochondrial functions. Of which, superparamagnetic iron oxide (SPIO)-NPs or graphene oxide-quantum dots (GO-QDs) was selected as representatives for positive or negative inducer of mitophagy. SPIO-NPs, but not GO-QDs, activated PINK1-dependent mitophagy as demonstrated by recruitment of PARKIN to mitochondria and degradation of injured mitochondria. SPIO-NPs caused the loss of mitochondrial membrane potential, decrease in ATP, and increase in mitochondrial reactive oxide species and Ca²⁺. Blocking mitophagy with PARKIN siRNA aggravated the cytotoxicity of SPIO-NPs. Taken together, PINK1 immunofluorescence-based HCA is considered to be an early, sensitive, and reliable approach to evaluate the bioimpacts of NPs.     

Small interfering RNA directed against CTMP reduces acute traumatic brain injury in a mouse model by activating Akt

Abstract

Objective:

Protein kinase B (PKB/Akt), which is phosphorylated and activated by upstream activators, exerts critical neuroprotective effects by phosphorylating downstream targets after traumatic brain injury (TBI). Studies on the regulation of Akt will be crucial for our understanding of neuronal survival. The goal of this study is to investigate the effects of carboxyl-terminal modulator protein (CTMP) on phosphorylation of Akt and neurological function in a mouse model of TBI.

Methods:

Traumatic brain injury in mice was performed by a controlled cortical impact device. The expression of Akt, phospho-Akt, and CTMP was examined in the injured cortices by immunohistochemistry and Western blot analysis. To determine the effects of CTMP, small interfering RNAs (siRNAs) directed against CTMP were injected in mice with TBI, and the expression of phosphorylated Akt and neurological function were evaluated.

Results:

Phospho-Akt significantly increased at 4 hours post-TBI in the nucleus (P < 0·01) and remained at high levels until 72 hours after TBI, as shown by Western blot analysis. In the cytosol, the expression of phospho-Akt reached its peak at 4 hours post-TBI, but decreased markedly at 24 hours and maintained below pre-TBI levels until 72 hours post-TBI. Interestingly, the expression of CTMP significantly increased 4 hours after TBI (P < 0·01) and sustained those levels until 72 hours without dramatic changes. Treatment with CTMP siRNA effectively augmented the phosphorylation of Akt and significantly improved the neurological functional recovery up to 28 days post-TBI.

Conclusion:

We conclude that Akt is phosphorylated and translocated to nucleus after TBI to exert neuroprotective effects. However, CTMP is simultaneously triggered to inhibit the phosphorylation of Akt. Inhibition of CTMP by siRNA improves the recovery of neurological functions after TBI.     

SiRNA联合介导Livin和Survivin基因沉默诱导人肾癌细胞786-O化疗敏感性的实验研究

目的:观察siRNA联合介导Livin和Survivin基因的沉默诱导人肾癌细胞786-O化疗敏感性的作用。方法:构建Livin和Suvivin联合靶向的小干扰RNA(siRNA)重组表达载体shRNA,然后将目的载体转染于人肾癌细胞株786-O。应用实时荧光定量聚合酶链反应(Real-time PCR)及Western blot方法分别检测转染前细胞经顺铂、5-FU和丝裂霉素处理后的Livin和Survivin基因的mRNA与蛋白水平的变化。应用CCK-8试验检测转染前后细胞对顺铂、5-氟尿嘧啶(5-FU)和丝裂霉素的半数致死量(IC50)、细胞增殖的变化。结果:CCK-8结果显示,联合介导Livin和Survivin基因沉默组细胞较分别单独介导细胞组化疗的敏感性明显增强(P0.05),且转染前后细胞对顺铂、5-FU和丝裂霉素的IC50发生显著变化(P0.05)。结论:SiRNA联合介导Livin和Survivin基因表达下调,发挥协同增强的siRNA作用。