Coumarin-Based Noncytotoxicity Fluorescent Dye for Tracking Actin Protein in In-Vivo Imaging.

Drosophila shares maximum homology with the human disease-causing genes and thus has been employed to evaluate the toxicity of numerous compounds. Further, its distinguishable developmental stages, easy rearing, and short lifespan make it a perfect model organism to study toxicological properties of any new compound. The current study evaluates the toxic effect of a coumarin-based organic fluorescent dye, 7-hydroxy-4-methyl-8-((4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl)-2H-chromen-2-one (CTC), using Drosophila melanogaster as a model organism by studying different behavioral, screening, and staining techniques using Oregon-R flies. For toxicity assessment, one control fly group was compared with various flies that had been subjected to fed CTC dye orally of different concentrations (0.5, 1, 2.5, and 5 µg/mL). The 3rd instar larvae were checked for the larvae crawling assay. The crawling assay demonstrates that the speed and path of the treated larvae are almost equal to the control ones, which signifies the non-neurotoxic property of CTC. Trypan blue assay further suggested that the dye does not cause any major damage to the gut. Phalloidin staining revealed that the actin composition remains unaltered even after the CTC treatment, while the DAPI staining experiment indicates that CTC does not cause any nuclear damage to fly gut cells. However, at a concentration of 5 µg/mL, CTC causes developmental delay. The flies hatched after larval treatment of CTC do not show any structural defects, suggesting clearly that CTC is also nongenotoxic to Drosophila. The current studies propose CTC as a noncytotoxic and nongenotoxic dye to track actin protein in the model organism D. melanogaster.

Synthesis of First Coumarin Fluorescent Dye for Actin Visualization.

Selective fluorescence imaging of actin protein hugely depends on the fluorescently labeled actin-binding domain (ABD). Thus, it is always a challenging task to image the actin protein (in vivo or in vitro) directly with an ABD-free system. To overcome the limitations of actin imaging without an ABD, we have designed a facile and cost-effective red fluorescent coumarin dye 7-hydroxy-4-methyl-8-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl-2H-chromen-2-one (CTC) for actin binding. The selective binding of the dye was investigated using the gut and eye of the model organism Drosophila melanogaster and C2C12 and SCC-9 cell lines. Our result suggests two major advantages of CTC over the dyes presently used for imaging actin proteins. First, the dye can bind to actin efficiently without any secondary intermediate. Second, it is much more stable at room temperature and exhibits excellent photostability. To the best of our knowledge, this is the first fluorescent dye that can bind to the actin protein without employing any secondary intermediate/actin-binding domain. These findings could pave the way for many biologists and physicists to successfully employ the CTC dye for imaging and tracking actin proteins by fluorescence microscopy in various in vivo and in vitro systems.

Environmental cue affects the hearing-related behaviors of Drosophila melanogaster by targeting the redox pathways.

Environmental cues like noise, pressure, and circadian rhythm can affect the hearing ability of human beings. Nevertheless, the complex physiology of the human being does not allow us to understand how these factors can affect hearing and hearing-related behaviors. Conversely, these effects can be easily checked using the hearing organ of Drosophila melanogaster, the Johnston organ. In the current study, the Drosophila was exposed to challenging environments like noise, low pressure, and altered circadian rhythm. The hearing organ of larvae, as well as adults, was analyzed for hearing-related defects. In the third instar larva, the cell deaths were detected in the antenna imaginal disc, the precursor of Johnston's organ. Elevated levels of reactive oxygen species and antioxidant enzymes were also detected in the adult antennae of environmentally challenged flies. The ultrastructure of the antennae suggests the presence of abundant mitochondria in the scolopidia of control. Fewer amounts of mitochondria are found in the environmentally challenged adult antennae. In adults, various hearing-related behaviors were analyzed as a readout of functionality of the hearing organ. Analysis of climbing, aggressive, and courtship behaviors suggests abnormal behavior in environmentally challenged flies than the control. The current study suggests that the environmental cues can alter hearing-related behaviors in Drosophila. The methods used in this study can be used to monitor the environmental pollution or to study the effect of alteration of noise, pressure, and circadian rhythm on hearing-related behaviors taking Drosophila melanogaster as a model organism. Graphical abstract.

Fe3O4 coated guargum nanoparticles as non-genotoxic materials for biological application.

The current study aims to check various behavioural, developmental, cytotoxic, and genotoxic effects of Fe3O4-GG nanocomposite (GGNCs) on Drosophila melanogaster. Fe3O4 nanoparticles were prepared by the chemical co-precipitation method and cross-linked with guargum nanoparticles to prepare the nanocomposites. The nanocomposites were characterized by using transmission electron microscopy (TEM), X-ray diffraction (XRD), and FTIR techniques. To investigate the biomolecular interaction, GGNCs was further tagged with Fluorescein isothiocyanate. Various concentrations of nanocomposites were mixed with the food and flies were allowed to complete the life cycle. The life cycle of the flies was studied as a function of various concentrations of GGNCs. The 1st instar larvae after hatching from the egg start eating the food mixed with GGNCs. The 3rd instar larvae were investigated for various behavioural and morphological abnormalities within the gut. The 3rd instar larva has defective crawling speed, crawling path, and more number of micronuclei within the gut. Similarly, in adult flies thermal sensitivity, climbing behaviour was found to be altered. In adult flies, a significant reduction in body weight was found which is further correlated with variation of protein, carbohydrate, triglyceride, and antioxidant enzymes. Altogether, the current study suggests GGNCs as a non-genotoxic nanoparticle for various biological applications.

Assessing the potential of lignin nanoparticles as drug carrier: Synthesis, cytotoxicity and genotoxicity studies.

Lignin nanoparticles synthesis is among recent developments in lignin valorization especially for biomedical applications. In this study, a new technique where complete self-assembling of lignin was ensured by simultaneous solvent displacement and flash pH change was used to optimize particle size of blank lignin nanoparticles (BLNPs) for suitability in cell uptake along with maximized yield. To establish BLNPs as drug carrier, safety studies including hemocompatibility, cytotoxicity and elaborate genotoxicity studies on Drosophila melanogaster as a model organism were done. Finally, irinotecan loaded lignin nanoparticles (DLNPs) were synthesized to establish their drug carrying potential and thorough in vitro characterization was performed. BLNPs with controllable size (⁓152 nm), low polydispersity (<0.2), maximized yield (>65%), negative surface charge (-22 to -23 mV), spherical shape and smooth surface were obtained with acceptable %hemolysis (<2%). In vitro cytotoxicity studies revealed that BLNPs were significantly toxic (74.38 ± 4.74%) in human breast adenocarcinoma (MCF-7), slightly toxic (38.8 ± 4.70%) in human alveolar epithelial adenocarcinoma (A-549) and insignificantly toxic (15.89 ± 2.84%) to human embryonic kidney (HEK-293) cells. BLNPs showed concentration dependent early neuronal defects in Drosophila, but nuclei fragmentation and gut cell damage were absent. Sustained release DLNPs with high drug loading reduced the IC50 value of irinotecan by almost 3 folds.