Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNA-mediated gene silencing

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through the RNA interference (RNAi) mechanism. Here, live-cell and live-animal bioluminescent imaging (BLI) is used to directly compare luciferase knockdown by unmodified and nuclease-stabilized siRNAs in rapidly (HeLa) and slowly (CCD-1074Sk) dividing cells to reveal the impact of cell division and siRNA nuclease stability on the kinetics of siRNA-mediated gene silencing. Luciferase knockdown using unmodified siRNAs lasts approximately 1 week in HeLa cells and up to 1 month in CCD-1074Sk cells. There is a slight increase in the duration of luciferase knockdown by nuclease-stabilized siRNAs relative to unmodified siRNAs after cationic lipid transfection, but this difference is not observed after electroporation. In BALB/cJ mice, a fourfold increase in maximum luciferase knockdown is observed after hydrodynamic injection (HDI) of nuclease-stabilized siRNAs relative to unmodified siRNAs, yet the overall kinetics of the recovery after knockdown are nearly identical. By using a mathematical model of siRNA-mediated gene silencing, the trends observed in the experimental data can be duplicated by changing model parameters that affect the stability of the siRNAs before they reach the cytosolic compartment. Based on these findings, we hypothesize that the stabilization advantages of nuclease-stabilized siRNAs originate primarily from effects prior to and during internalization before the siRNAs can interact with the intracellular RNAi machinery.     

A modified static respiration assay and its relationship with an enzymatic test to assess compost stability and maturity

Despite the numerous compost stability and maturity tests, no universally accepted compost stability or maturity index exists. The fluorescein di-acetate (FDA) enzymatic assay, originating from soil studies, is examined here as a potential new compost stability test, and is compared to microbial respiration and phytotoxicity indices. Thirteen composts were used in the study from different source materials. Static microbial respiration activity indices calculated were the cumulative O₂ consumptions, O₂ consumption rates, total C-CO₂ production, the respiratory quotient and the bio C/N ratio. Compost phytotoxicity was quantified via a 7-day tomato seed germination assay. Results showed that the net fluorescein release rates correlated with all stability indices. The germination index marginally correlated with the fluorescein release rates, but not with any of the other stability indices. New limits to classify composts regarding their stability were proposed.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

RNAi介导的棉铃虫氨肽酶N基因Haapn1和钙粘蛋白基因Ha_BtR沉默对Cry1Ac毒力的影响

氨肽酶N（aminopeptidase N, APN）和钙粘蛋白（cadherin）是存在于鳞翅目昆虫中肠刷状缘膜囊（brush border membrane vesicles, BBMV）上Bt毒素Cry1A的受体。本实验将棉铃虫Helicoverpa armigera氨肽酶N1基因Haapn1和钙粘蛋白基因Ha_BtR双链RNA（dsRNA）注入棉铃虫4龄幼虫体内, 以研究这两种受体基因沉默后对Cry1Ac毒力的影响。结果表明: 注射dsRNA（1 μg/头）进行基因沉默后, Haapn1 mRNA表达量比注射缓冲液（elution solution, ES）的对照下降了30%~49%, Ha_BtR mRNA表达量下降了30%~37%。注射Haapn1 dsRNA的幼虫在40和70 μg/cm² Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫, 而在 100 和 170 μg/cm² Cry1Ac原毒素处理下两者死亡率无显著差异; Cry1Ac活化毒素以及原毒素对注射Ha_BtR dsRNA幼虫与注射ES幼虫的毒力均无显著差异。当同时注射Haapn1及Ha_BtR dsRNA后, 干扰后的幼虫对Cry1Ac活化毒素和原毒素的敏感性均显著下降。本研究进一步证明了棉铃虫Haapn1和Ha_BtR均是Bt毒素Cry1Ac的功能受体, 这两种受体蛋白共同参与Cry1Ac的毒杀作用过程。该结果也提示, Haapn1或Ha_BtR基因产生突变都可能导致棉铃虫对Cry1Ac产生抗性。     

Novel whey-derived peptides with inhibitory effect against angiotensin-converting enzyme: in vitro effect and stability to gastrointestinal enzymes

Whey protein concentrate (WPC) was subjected to enzymatic hydrolysis by proteases from the flowers of Cynara cardunculus, and the resulting angiotensin-converting enzyme (ACE)-inhibitory effect was monitored. The whole WPC hydrolysate exhibited an IC₅₀ value of 52.9 ± 2.9 μg/mL, whereas the associated peptide fraction with molecular weight below 3 kDa scored 23.6 ± 1.1 μg/mL. The latter fraction was submitted to RP-HPLC, and 6 fractions were resolved that exhibited ACE-inhibitory effects. Among the various peptides found, a total of 14 were identified via sequencing with an ion-trap mass spectrometer. Eleven of these peptides were synthesized de novo - to validate their ACE-inhibitory effect, and also to ascertain their stability when exposed to simulated gastrointestinal digestion. Among them, three novel, highly potent peptides were found, corresponding to α-lactalbumin f(16-26) - with the sequence KGYGGVSLPEW, α-lactalbumin f(97-104) with DKVGINYW, and β-lactoglobulin f(33-42) with DAQSAPLRVY; their IC₅₀ values were as low as 0.80 ± 0.1, 25.2 ± 1.0 and 13.0 ± 1.0 μg/mL, respectively. None of them remained stable in the presence of gastrointestinal enzymes: they were partially, or even totally hydrolyzed to smaller peptides - yet the observed ACE-inhibitory effects were not severely affected for two of those peptides.     

Use of the modified viral satellite DNA vector to silence mineral nutrition-related genes in plants: silencing of the tomato ferric chelate reductase gene, FRO1, as an example

Virus-induced gene silencing (VIGS) is potentially an attractive reverse-genetics tool for studies of plant gene function, but whether it is effective in silencing mineral nutritional-related genes in roots has not been demonstrated. Here we report on an efficient VIGS system that functions in tomato roots using a modified viral satellite DNA (DNAmβ) associated with Tomato yellow leaf curl China virus (TYLCCNV). A cDNA fragment of the ferric chelate reductase gene (FRO1) from tomato was inserted into the DNAmβ vector. Tomato roots agro-inoculated with DNAmβ carrying both a fragment of FRO1 and TYLCCNV used as a helper virus exhibited a significant reduction at the FRO1 mRNA level. As a consequence, ferric chelate reductase activity, as determined by visualization of the pink FeBPDS3 complex was significantly decreased. Our results clearly demonstrated that VIGS system can be employed to investigate gene function associated with plant nutrient uptake in roots.     

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H₂O₂) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H₂O₂ treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H₂O₂ was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H₂O₂ induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer.     

Combined inhibition of Chk1 and Wee1: In vitro synergistic effect translates to tumor growth inhibition in vivo

Targeting Chk1 protein kinase can enhance the antitumor effects of radio- and chemotherapy. Recent evidence disclosed a role of Chk1 in unperturbed cell proliferation and survival, implying that Chk1 inhibitors could also be effective as single agents in tumors with a specific genetic background. To identify genes in synthetic lethality with Chk1, we did a high-throughput screening using a siRNA library directed against 719 human protein kinases in the human ovarian cancer cell line OVCAR-5, resistant to Chk1 inhibitors. Wee1 tyrosine kinase was the most significant gene in synthetic lethality with Chk1. Treatment with non-toxic concentrations of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) confirmed the marked synergistic effect in various human cancer cell lines (breast, ovarian, colon, prostate), independently of the p53 status. Detailed molecular analysis showed that the combination caused cancer cells to undergo premature mitosis before the end of DNA replication, with damaged DNA leading to cell death partly by apoptosis. In vivo treatment of mice bearing OVCAR-5 xenografts with the combination of Chk1 and Wee1 inhibitors led to greater tumor growth inhibition than with the inhibitors used as single agents with no toxicity. These data provide a strong rationale for the clinical investigation of the combination of a Chk1 and a Wee1 inhibitor.     

The effect of budesonide/formoterol maintenance and reliever therapy on the risk of severe asthma exacerbations following episodes of high reliever use: an exploratory analysis of two randomised,controlled studies with comparisons to standard therapy

Background

Divergent strategies have emerged for the management of severe asthma. One strategy utilises high and fixed doses of maintenance treatment, usually inhaled corticosteroid/long-acting β₂-agonist (ICS/LABA), supplemented by a short-acting β₂-agonist (SABA) as needed. Alternatively, budesonide/formoterol is used as both maintenance and reliever therapy. The latter is superior to fixed-dose treatment in reducing severe exacerbations while achieving similar or better asthma control in other regards. Exacerbations may be reduced by the use of budesonide/formoterol as reliever medication during periods of unstable asthma. We examined the risk of a severe exacerbation in the period after a single day with high reliever use.

Methods

Episodes of high reliever use were quantified and exacerbations occurring post-index day with these episodes were examined post hoc in two double-blind studies comparing the efficacy and safety of budesonide/formoterol maintenance and reliever therapy (Symbicort SMART™, Turbuhaler®) 160/4.5 μg twice daily plus as needed with similar or higher maintenance doses of ICS/LABA plus SABA or formoterol.

Results

Budesonide/formoterol maintenance and reliever therapy significantly reduced the risk of episodes of high reliever use (>6 inhalations/day) vs. all alternative ICS/LABA regimens. With conventional fixed-dose treatment the need for exacerbation treatment within 21 days ranged from 6.0–10.1% of days post-index for all regimens compared with 2.5–3.4% of days with budesonide/formoterol maintenance and reliever therapy.

Conclusions

Budesonide/formoterol maintenance and reliever therapy reduces the incidence of high reliever episodes and the exacerbation burden immediately following these episodes vs. alternative ICS/LABA plus SABA regimens at up to double the maintenance dose of ICS.

Trial registration

These studies do not have registration numbers as they were conducted before clinical trial registration was required