TREX reveals proteins that bind to specific RNA regions in living cells.

Different regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells and under endogenous settings. Here we introduce TREX (targeted RNase H-mediated extraction of crosslinked RBPs)-a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD long noncoding RNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and previously unknown interactions that regulate ribosome biogenesis. With its applicability to different cell types, TREX is an RNA-centric tool for unbiased positional mapping of endogenous RNA-protein interactions in living cells.

Comparative transcriptome and proteome analysis to reveal the biosynthesis of gold nanoparticles in Arabidopsis.

A large number of plants have been tested and exploited in search of a green chemistry approach for the fabrication of gold or other precious metal nanomaterials. Despite the potential of plant based methods, very little is known about the underlying biochemical reactions and genes involved in the biotransformation mechanism of AuCl4 into gold nanoparticles (AuNPs). In this research, we thus focused on studying the effect of Au on growth and nanoparticles formation by analyses of transcriptome, proteome and ionome shift in Arabidopsis. Au exposure favored the growth of Arabidopsis seedling and induced formation of nanoparticles in root and shoot, as indicated by optical and hyperspectral imaging. Root transcriptome analysis demonstrated the differential expression of the members of WRKY, MYB and BHLH gene families, which are involved in the Fe and other essential metals homeostasis. The proteome analysis revealed that Glutathione S-transferases were induced in the shoot and suggested its potential role in the biosynthesis AuNPs. This study also demonstrated the role of plant hormone auxin in determining the Au induced root system architecture. This is the first study using an integrated approach to understand the in planta biotransformation of KAuCl4 into AuNPs.

Microarray analysis of Arabidopsis under gold exposure to identify putative genes involved in the synthesis of gold nanoparticles (AuNPs).

Very little is known about the genes responsible for Au uptake, reduction and detoxification in plants, which indeed essential to understand the complex trait of AuNP biosynthesis. We designed a targeted experiment to elucidate the response of plant at transcriptional level under Au exposure, and a microarray was performed on root tissue treated with AuCl4 (-) in the absence of nutrient media to record specific gene expression signature. Here, we describe the experimental procedures and data analysis in detail to reproduce the results (available at GEO database under GSE55436) published by Shukla et al. (2014) [1] in the Frontiers in Plant Sciences. The data produced from this study provide significant information of genes which may be used to enhance the AuNP biosynthesis.

Facile Synthesis of Monodisperse Pt and Pd Nanoparticles Using Antioxidants.

Monodispersed Pt and Pd nanoparticles (NPs) were synthesized using the antioxidant gallic acid as a reducing and capping agent. The effects of the reaction time and gallic acid concentration on reduction of metal ions were studied and it was found that these were critical factors affecting the size, shape and productivity of Pt and Pd NPs. Transmission electron microscopy (TEM) analysis showed the presence of spherical NPs of 1 to 30 nm at different reaction times. It was observed that the size of Pt and Pd NPs increased with respect to reaction time. The Pt and Pd NPs with average size of 21 nm were well dispersed after the reaction completed. X-ray diffraction (XRD) and selective area electron diffraction (SAED) revealed the formation of crystalline Pt and Pd NPs. The diffraction peaks at 39 degrees, 45 degrees, 66 degrees and 80 degrees, corresponding to the (111), (200), (220) and (311) lattice planes, respectively, were observed in the Pt and Pd NPs. Inductively coupled plasma (ICP) analysis for residual concentrations of Pt and Pd ions showed that 100% conversion was achieved within 72 h of reaction. Hence, the use of antioxidant as a reducing and capping agent could be an alternative to the conventional synthesis method. This environmentally-benign, simple and cost effective method of synthesizing Pt and Pd NPs may be a novel electrode material for use in fuel cell.

Genome wide transcriptome analysis reveals ABA mediated response in Arabidopsis during gold (AuCl(-) 4) treatment.

The unique physico-chemical properties of gold nanoparticles (AuNPs) find manifold applications in diagnostics, medicine and catalysis. Chemical synthesis produces reactive AuNPs and generates hazardous by-products. Alternatively, plants can be utilized to produce AuNPs in an eco-friendly manner. To better control the biosynthesis of AuNPs, we need to first understand the detailed molecular response induced by AuCl(-) 4 In this study, we carried out global transcriptome analysis in root tissue of Arabidopsis grown for 12- h in presence of gold solution (HAuCl4) using the novel unbiased Affymetrix exon array. Transcriptomics analysis revealed differential regulation of a total of 704 genes and 4900 exons. Of these, 492 and 212 genes were up- and downregulated, respectively. The validation of the expressed key genes, such as glutathione-S-transferases, auxin responsive genes, cytochrome P450 82C2, methyl transferases, transducin (G protein beta subunit), ERF transcription factor, ABC, and MATE transporters, was carried out through quantitative RT-PCR. These key genes demonstrated specific induction under AuCl4(-) treatment relative to other heavy metals, suggesting a unique plant-gold interaction. GO enrichment analysis reveals the upregulation of processes like oxidative stress, glutathione binding, metal binding, transport, and plant hormonal responses. Changes predicted in biochemical pathways indicated major modulation in glutathione mediated detoxification, flavones and derivatives, and plant hormone biosynthesis. Motif search analysis identified a highly significant enriched motif, ACGT, which is an abscisic acid responsive core element (ABRE), suggesting the possibility of ABA- mediated signaling. Identification of abscisic acid response element (ABRE) points to the operation of a predominant signaling mechanism in response to AuCl(-) 4 exposure. Overall, this study presents a useful picture of plant-gold interaction with an identification of candidate genes involved in nanogold synthesis.

Yucca-derived synthesis of gold nanomaterial and their catalytic potential.

AuNPs ranging in 20 to 300 nm size were synthesized at a room temperature using Yucca filamentosa leaf extract. Diverse nanomaterial morphologies were obtained by varying the extract concentration, reaction pH, and temperature. While low volumes of extract (0.25 and 0.5 mL) induced the formation of microscale Au sheets with edge length greater than 1 µm, high volumes yielded spherical particles ranging from 20 to 200 nm. Varying pH of the solution significantly influenced the particle shape with the production of largely spherical particles at pH 5 to 6 and truncated triangles at pH 2. Separation of multidimensional nanostructures was achieved using a novel method of sucrose density gradient centrifugation. The catalytic function of Yucca-derived AuNPs was demonstrated by degradation of a wastewater dye: methylene blue using spectrophotometric measurements over time. Treatment with Au nanosheets and spheres demonstrated methylene blue degradation approximately 100% greater than the activity in control at 60 min.

Role of Fe-responsive genes in bioreduction and transport of ionic gold to roots of Arabidopsis thaliana during synthesis of gold nanoparticles.

Several studies have shown potassium chloroaurate (KAuCl4)-mediated synthesis of gold nanoparticles (AuNPs) by using extracts of different parts of diverse plant species. However, the mechanism underlying the formation of AuNPs in planta has far from being elucidated. Here, we report the molecular evidence towards the role of genes involved in iron (Fe) homeostasis during in planta synthesis of AuNPs in roots of Arabidopsis thaliana. Firstly, we examined the dosage-dependent effects of KAuCl4 treatment on primary root length (PRL), and meristematic activity of roots in transgenic CycB1;1::uidA. Compared to control seedling (0 ppm KAuCl4), PRL and meristematic activity of primary and lateral roots showed progressive attenuation in seedlings treated with higher concentrations of KAuCl4 (25 ppm or above). Therefore, subsequent studies on in planta synthesis of AuNPs, and molecular responses were carried out in roots of the seedlings treated with 10 ppm KAuCl4 for 7 d. TEM of KAuCl4-treated seedlings showed the presence of monodisperse AuNPs of different shapes and sizes in root biomatrix. There was a significant induction of FRO2 in KAuCl4-treated roots, and therefore its likely involvement in bioreduction of Au(3)(+) could be assumed. Elevated expression levels of Fe transporters IRT1 and IRT2 further suggested their potential role in transport of bioreduced Au(3+) across root membrane. Expression levels of other genes involved in Fe homeostasis, and also different members of zinc (Zn), phosphate (Pi), and potassium (K) transporter families remained unaffected by KAuCl4 treatment. An increased Au content in Fe-deprived roots further provided evidence towards the specific role of a subset of Fe-responsive genes during in planta synthesis of AuNPs.