Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana.

Advanced transcriptome sequencing has revealed that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the model plant Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionarily conserved transcripts, PIN7a and PIN7b. PIN7a and PIN7b, differing in a four amino acid stretch, exhibit almost identical expression patterns and subcellular localization. We reveal that they are closely associated and mutually influence each other's mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, revealing an additional regulatory level of auxin-mediated plant development.

Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila.

ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.

Green synthesis of anisotropic gold nanoparticles for photothermal therapy of cancer.

Nanoparticles of varying composition, size, shape, and architecture have been explored for use as photothermal agents in the field of cancer nanomedicine. Among them, gold nanoparticles provide a simple platform for thermal ablation owing to its biocompatibility in vivo. However, the synthesis of such gold nanoparticles exhibiting suitable properties for photothermal activity involves cumbersome routes using toxic chemicals as capping agents, which can cause concerns in vivo. Herein, gold nanoparticles, synthesized using green chemistry routes possessing near-infrared (NIR) absorbance facilitating photothermal therapy, would be a viable alternative. In this study, anisotropic gold nanoparticles were synthesized using an aqueous route with cocoa extract which served both as a reducing and stabilizing agent. The as-prepared gold nanoparticles were subjected to density gradient centrifugation to maximize its NIR absorption in the wavelength range of 800-1000 nm. The particles also showed good biocompatibility when tested in vitro using A431, MDA-MB231, L929, and NIH-3T3 cell lines up to concentrations of 200 µg/mL. Cell death induced in epidermoid carcinoma A431 cells upon irradiation with a femtosecond laser at 800 nm at a low power density of 6 W/cm(2) proved the suitability of green synthesized NIR absorbing anisotropic gold nanoparticles for photothermal ablation of cancer cells. These gold nanoparticles also showed good X-ray contrast when tested using computed tomography (CT), proving their feasibility for use as a contrast agent as well. This is the first report on green synthesized anisotropic and cytocompatible gold nanoparticles without any capping agents and their suitability for photothermal therapy.

Ambient temperature synthesis of citrate stabilized and biofunctionalized, fluorescent calcium fluoride nanocrystals for targeted labeling of cancer cells.

Targeted biological contrast agents are emerging as promising candidates in the field of cancer theragnostics. Herein, we report an ambient temperature synthesis of a nanosized, antibody functionalized lanthanide doped CaF2 biolabel and demonstrate in vitro its potential for cancer cell targeting efficacy and specificity. Monodispersed citrate stabilized lanthanide (Eu3+) doped CaF2 nanoparticles with size ∼25 nm, exhibiting strong fluorescent emission at 612 nm, were prepared using an aqueous wet chemical route at room temperature. Biofunctionalization of the fluorescent nanoparticles using an anti-EGFR antibody through EDC-NHS coupling chemistry enabled targeting of EGFR over-expressing cells. The nanobioconjugates showed preferential binding to EGFR+ve oral epithelial carcinoma cells (KB) and human epidermoid carcinoma cells (A431) with no accumulation onto EGFR-ve non-cancerous NIH 3T3 cells. The fluorescence was maintained after the bioconjugation as well as after attachment to the cancer cells, demonstrating their potential as targeted biolabels. Cytotoxicity evaluation with several cancerous (A431, KB) and non-cancerous (NIH 3T3, L929) cell lines revealed no toxicity at concentrations up to 1 mM. Thus, the fluorescence characteristics and biocompatibility, coupled with the molecular receptor targeting capability, suggest the potential use of CaF2 in the field of bioimaging.

In vitro targeted imaging and delivery of camptothecin using cetuximab-conjugated multifunctional PLGA-ZnS nanoparticles.

BACKGROUND: Targeted cancer therapy has been extensively developed to improve the quality of treatment by reducing the systemic exposure of cytotoxic drug. Polymeric nanoparticles with conjugated targeting agents are widely investigated because they offer tunability in particle size, drug release profile and biocompatibility. MATERIALS & METHODS: Here, we have prepared targeted multifunctional nanoparticles composed of a poly(lactic-co-glycolic acid) matrix, ZnS:Mn(2+) quantum dots and camptothecin, and targeted them to EGF receptor overexpressing cells with a cetuximab antibody. RESULTS: Physicochemical characterization of multifunctional nanoparticles showed stable particles with sizes of <200 nm. In vitro drug release and blood contact studies showed a sustained release profile, with limited hemolysis. In vitro cytotoxicity and cell uptake studies were carried out in A549, KB and MFC-7 cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, FACS, fluorescent microscopic images and spectroflourimetry. CONCLUSION: Our studies revealed higher camptothecin activity and uptake in cell lines that overexpress the EGF receptor. All these results suggest that anti-EGF receptor cetuximab-conjugated poly(lactic-co-glycolic acid) multifunctional nanoparticles can be used as a potential nanomedicine against cancer.