Mapping RNA-protein interactions with subcellular resolution using colocalization CLIP.

RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP.

RNA binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the well-studied RNA-binding protein HuR. Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuR's unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

A flow cytometric method to assess nanoparticle uptake in bacteria.

Toxicity of engineered nanomaterials (ENMs), such as metal oxides, has been of concern among environmental and health scientists. For ecotoxicity studies of ENMs, it is important to assess nanoparticle uptake and correlate it with the cellular response. However, due to nonavailability of adequate methods for assessing cellular uptake of ENMs, there is a lack of information in this important area. In the present study, a method has been developed using flow cytometry, which allows for rapid detection of ENM internalization in live bacteria under different experimental conditions for several generations. Our data demonstrate significant internalization of Zinc oxide (ZnO) and Titanium (IV) oxide (TiO(2) ) nanoparticles (NPs) in Escherichia coli in a dose-dependent manner. ZnO NPs treatment exhibited a significant increase in the intensity of side scatter (SSC) with liver-S9 fraction (76, 94, and 181% increase) rather than without S9 (10.5, 24.5, and 125.9% increase) at 10, 40, and 80 µg/ml concentrations, respectively. This was due to the protein coating of NPs by the S9 fraction. A similar response was also observed on exposure to TiO(2) NPs (139 and 203% with S9 and 128 and 198% without S9). In a multigeneration study, this new method was able to detect the presence of ENMs in E. coli up to four generations. Our data demonstrate that this method can be used for assessing the uptake of ENMs in bacteria and provides a handle to toxicologists for ecotoxicity studies of economically important ENMs to ensure safer products in the market.

Evidence for natural vertical transmission of chikungunya viruses in field populations of Aedes aegypti in Delhi and Haryana states in India-a preliminary report.

Aedes aegypti and Aedes albopictus are principal vectors for the transmission of chikungunya virus (CHIKV). India is a hub for both dengue and chikungunya infections and there are several reports of co-infection of dengue and chikungunya virus in the clinical scenario. The present pilot entomological survey was conducted to evaluate vertical transmission of CHIKV in Aedes field populations. Aedes immature (larvae and pupae) collection was done in 2012, over a period of six months from selected sites in Delhi and Haryana, India. The immatures collected were reared for adult emergence and species identification was done. A. aegypti male and female mosquitoes were separated and pooled collection spot-wise, RNA extracted and RT PCR performed to test for the presence of CHIKV in the pools. Container index (CI) and minimum infection rate (MIR) were estimated. From study areas that tested positive for CHIKV, adult collections were made and females upon feeding on uninfected blood in laboratory were allowed to lay eggs. The progeny that emerged from these field-collected mothers were tested for CHIKV presence. Our pilot survey showed the existence of A. aegypti population even during peak summer season in a few foci which eventually helped the mosquitoes to tide over adverse environmental conditions and with the start of rainfall, the population exploded within a short period of time. Immatures collected from field and progeny of adults collected from the field were CHIKV positive demonstrating the presence of vertical transmission of chikungunya virus in field population of A. aegypti. The present study further demonstrates the importance of identifying permanent breeding sites for proper Aedes species control.

Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells.

Extensive production and consumption of nanomaterials such as ZnO and TiO(2) has increased their release and disposal into the environment. The accumulation of nanoparticles (NPs) in ecosystem is likely to pose threat to non-specific targets such as bacteria. The present study explored the effect of ZnO and TiO(2) NPs in a model bacterium, Salmonella typhimurium. The uptake of ZnO and TiO(2) bare NPs in nano range without agglomeration was observed in S. typhimurium. TEM analysis demonstrated the internalization and uniform distribution of NPs inside the cells. Flow cytometry data also demonstrates that both ZnO and TiO(2) NPs were significantly internalized in the S. typhimurium cells in a concentration dependent manner. A significant increase in uptake was observed in the S. typhimurium treated even with 8 and 80 ng mL(-1) of ZnO and TiO(2) NPs with S9 after 60 min, possibly the formation of micelles or protein coat facilitated entry of NPs. These NPs exhibited weak mutagenic potential in S. typhimurium strains TA98, TA1537 and Escherichia coli (WP2uvrA) of Ames test underscoring the possible carcinogenic potential similar to certain mutagenic chemicals. Our study reiterates the need for re-evaluating environmental toxicity of ZnO and TiO(2) NPs presumably considered safe in environment.

Engineered ZnO and TiO(2) nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli.

Extensive use of engineered nanoparticle (ENP)-based consumer products and their release into the environment have raised a global concern pertaining to their adverse effects on human and environmental health. The safe production and use of ENPs requires improvement in our understanding of environmental impact and possible ecotoxicity. This study explores the toxicity mechanism of ZnO and TiO(2) ENPs in a gram-negative bacterium, Escherichia coli. Internalization and uniform distribution of characterized bare ENPs in the nano range without agglomeration was observed in E. coli by electron microscopy and flow cytometry. Our data showed a statistically significant concentration-dependent decrease in E. coli cell viability by both conventional plate count method and flow cytometric live-dead discrimination assay. Significant (p<0.05) DNA damage in E. coli cells was also observed after ENP treatment. Glutathione depletion with a concomitant increase in hydroperoxide ions, malondialdehyde levels, reactive oxygen species, and lactate dehydrogenase activity demonstrates that ZnO and TiO(2) ENPs induce oxidative stress leading to genotoxicity and cytotoxicity in E. coli. Our study substantiates the need for reassessment of the safety/toxicity of metal oxide ENPs.

Titanium dioxide nanoparticles induce oxidative stress-mediated apoptosis in human keratinocyte cells.

Titanium dioxide nanoparticles (TiO2 NPs) are the most commonly used metal oxide NPs in various industrial and commercial products. The present study has demonstrated a significant cellular uptake of TiO2 NPs in the human keratinocyte cells (HaCaT) using transmission electron microscopy and flow cytometry. The data exhibited a significant (p < 0.05) concentration dependent decrease in cell viability and glutathione with concomitant increase in lipid peroxidation and reactive oxygen species. The increased oxidative stress further leads to apoptosis after 48 h of exposure. Our study demonstrates oxidative stress mediated apoptosis in human keratinocyte cells exposed to TiO2 NPs.

Cellular response to metal oxide nanoparticles in bacteria.

The exponential growth in the production and consumption of engineered nanoparticles (ENPs) has raised concern about their environmental fate. ENPs accumulation in ecosystems is likely to pose threat to specific and non-specific targets. In this study a novel approach using flow cytometry was validated for detection of ENPs (ZnO and TiO2) uptake in live bacteria for several generations. These ENPs also induced frameshift mutation in S. typhimurium strains of Ames test, thus underscoring their possible carcinogenic potential.