Mycophenolic acid (MPA) modulates host cellular autophagy progression in sub genomic dengue virus-2 replicon cells.

Cellular autophagy (Macrophagy) is a self-degradative process, executed through the network of autophagy associated genes (ATGs) encoded proteins. Both in vitro and in vivo studies suggest that dengue virus (DENV) induces autophagy and supports the viral genome replication and translation. Therefore, the cellular autophagy induced by dengue virus can be a good target for antiviral drug development. The action of mycophenolic acid (MPA), a specific inhibitor of DENV replication, was investigated in the stable BHK-21/DENV2 replicon cells. The inhibition was mediated by enhanced degradation of autophagic substrates in stable BHK-21/DENV2 replicon cells as evidenced by a decrease in lapidated LC3 (LC3II) and p62 expression in the presence of MPA. In contrast, the results indicated that four gene sets, namely Transmembrane protein 74 (TMEM74), Unc-51-like kinase 2 (ULK2), Cathepsin D (CTSD) and Estrogen receptor 1 (ESR1) were upregulated in stable BHK-21/DENV2 replicon cells, due to the sustained dynamic replication of DENV2 genome. These ATGs involved in the pre-autophagosomal structure (PAS) formation, were suppressed in the presence MPA. Instead, MPA induced the expression of different set of autophagy genes such as ATG4, AKT1, APP, ATG16L1, ATG16L2, B2M and HPRT1. An enzyme involved in the nucleotide salvage pathway, HPRT1, was highly expressed in the presence of MPA. The study shows that DENV2 replication is dependent on PAS formation and is inhibited in the presence of MPA by enhancing the degradation of autophagic substrates and suppression of PAS formation. This study provides impetus in designing MPA analogues to effectively inhibit dengue viral replication.

RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi) based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. The conclusion drawn from this analysis is that RNAi will still occupy specific domains of biomedical research and clinical applications, under the current state of development of these technologies. However, further improvements in CRISPR/Cas9 based technology may ultimately enable it to dominate RNAi in the long term.

Suppression of different classes of somatic mutations in Arabidopsis by vir gene-expressing Agrobacterium strains.

BACKGROUND: Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional ß-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of blue spots visible in plants grown for a further two weeks, we estimated the mutation frequencies. RESULTS: For plants co-cultivated for 48 h with Agrobacterium, if the strain contained vir genes, then the rates of transversions, SHRs and FSMs (measured 2 weeks later) were lower than those of uninfected controls. In contrast, co-cultivation for 48 h with any of the Agrobacterium strains raised the transposition rates above control levels. The multiple time-point study showed that in seedlings co-cultivated with wild type Ach5, the reduced rates of transversions and SHRs after 48 h co-cultivation represent an apparent suppression of an earlier short-lived increase in mutation rates (peaking for plants co-cultivated for 3 h). An increase after 3 h co-cultivation was also seen for rates of transversions (but not SHR) in seedlings exposed to the strain lacking vir genes, oncogenes and T-DNA. However, the mutation rates in plants co-cultivated for longer times with this strain subsequently dropped below levels seen in uninfected controls, consistent with the results of the single time-point study. CONCLUSIONS: The rates of various classes of mutations that result from Agrobacterium infection depend upon the duration of infection and the type of pathogen derived factors (such as Vir proteins, oncoproteins or T-DNA) possessed by the strain. Strains with vir genes, including the type used for plant transformation, suppressed selected classes of somatic mutations. Our study also provides evidence of a pathogen that can at least partly counter the induction of mutations in an infected plant.

Loss of Function Genetic Screen Identifies ATM Kinase as a Positive Regulator of TLR3-Mediated NF-κB Activation.

TLR3, a major innate immune pattern recognition receptor of RNA viruses, triggers inflammatory response through the transcription factor NF-κB. However, a genome-wide understanding of the genes and mechanisms regulating TLR3-mediated NF-κB activation is incomplete. We herein report the results of a human genome-wide RNAi screen that identified 591 proteins regulating TLR3-mediated NF-κB response. Bioinformatics analysis revealed several signaling modules including linear ubiquitination assembly complex and mediator protein complex network as regulators of TLR3 signaling. We further characterized the kinase ATM as a previously unknown positive regulator of TLR3 signaling. TLR3 pathway stimulation induced ATM phosphorylation and promoted interaction of ATM with TAK1, NEMO, IKKα, and IKKß. Furthermore, ATM was determined to coordinate the assembly of NEMO with TAK1, IKKα, and IKKß during TLR3 signaling. This study provided a comprehensive understanding of TLR3-mediated inflammatory signaling regulation and established a role for ATM in innate immune response.

A slow, efficient and safe nanoplatform of tailored ZnS QD-mycophenolic acid conjugates for in vitro drug delivery against dengue virus 2 genome replication.

Dengue is a major health concern causing significant mortality, morbidity and economic loss. The development of anti-dengue viral drugs is challenging due to high toxicity, as well as off-target/side effects. We engineered size tuned ZnS QDs as a platform for the efficient delivery of mycophenolic acid (MPA) against dengue virus serotype 2 (DENV2) to evaluate the drug efficacy and toxicity using the DENV2 sub-genomic replicon system in BHK21 cells. The results indicate that the Selectivity Index 50 (SI50) of the ZnS QD-MPA conjugate was two orders higher than that of free MPA with lower cytotoxicity. The effect is attributed to the sustained release of MPA from ZnS QD-MPA. The conjugated MPA caused significant inhibition of the virus at the level of replication and viral protein translation. The study underpins the efficiency of the ZnS QD for the delivery of antiviral drugs against DENV2 with negligible toxicity and side effects.

Label Free, Nontoxic Cu-GSH NCs as a Nanoplatform for Cancer Cell Imaging and Subcellular pH Monitoring Modulated by a Specific Inhibitor: Bafilomycin A1.

Metal nanoparticles-based sensors invoked much research attention in the biomedical field, especially in applications involving live cell imaging and monitoring. Here, a simple cost-effective method is adopted to synthesize glutathione coated copper nanoclusters (Cu-GSH NCs) with strong bright red fluorescence (625 nm). The clusters were found to be containing five Cu(0) atoms complexed with one molecule of glutathione (GSH) as evidenced by MALDI-TOF MS analysis. The synthesized Cu-GSH NCs system responds linearly to the pH in the acidic and alkaline ranges with a high degree of in vitro pH reversibility, projecting its potential as a real time pH sensor. Higher intensity emission observed in acidic conditions can be exploited for its employability as cellular organelle markers. The imaging and sensing potential of Cu-GSH NCs in the live human adenocarcinoma cell line, the HeLa cells, was tested. The treatment of HeLa cells for 48 h imparted deep red fluorescence, owing to the lower level of intracellular pH in cancer cells. In contrast, the imaging using normal cell lines (L-132, lung epithelial cell line) showed significantly lower fluorescence intensity as compared to that of HeLa cells. The subcellular pH-dependent fluorescence emission of Cu-GSH NCs was further assessed by treating HeLa cells with proton pump (V-ATPase) inhibitor Bafilomycin A1, which increases the vesicular pH. Interestingly, the fluorescent intensity of HeLa cells decreases with increasing concentration of Bafilomycin A1 in the presence of Cu-GSH NCs, as evidenced by the fluorescence microscopic images and quantitative fluorescent output. Accordingly, the developed Cu-GSH NCs system can be employed as an efficient pH-based bioimaging probe for the detection of cancer cells with an implied potential for the label free subcellular organelle tracking and marking. Importantly, the Cu-GSH NCs can be used for live cell pH imaging owing to their high degree of reversibility in sensing of pH variation.

Cytotoxicity of nanoparticles - Are the size and shape only matters? or the media parameters too?: a study on band engineered ZnS nanoparticles and calculations based on equivolume stress model.

Size dependent cytotoxicity of ZnS nanoparticles (NPs) was investigated in Human embryonic kidney (HEK-293) cell lines by MTT assay. The cells were incubated with varying concentration of ZnS NPs of sizes 4 nm, 10 nm and 25 nm for 48 h under different (cell culture) media viscosity conditions. The results showed that the toxicity is decreased with the particle size while it is negatively correlated with the viscosity of the media. Theoretical calculations were performed, by assuming equivolume stress model and the same is explained with schematics. Similarly, the effect of particle size and shape on toxicity is explained based on the theoretical calculation of the stress. The calculations showed that out of the possible cellular entry mechanisms for the cubic or cage shaped NPs, the highest toxicity is predicted for the entry through the corners while the lowest toxicity is predicted for the entry through the faces. The experimental observations depicting the cytotoxicity as a function of the viscosity of cell culture media was also validated by stress calculations and are found to be consistent. Studies on size and shape dependence of semiconductor NPs like ZnS is rather scarce, while the role of viscosity of cell culture media on the cytotoxicity is being reported for the first time. In summary, the study indicates that the cytotoxicity is an integral function of size and shape of NPs, physical parameters of the cell culture media in addition to the post entry biochemical interactions with the host cell.