Gayatri Udayan

Nanoplastics pose significant environmental and public health risks, prompting the need for sensitive, cost-effective, and rapid assays for ecotoxicity assessment. The present work proposes the use of a portable smartphone-based platform to enhance traditional Daphnia magna acute toxicity assays by integrating behavior analysis and heart rate measurements. The aim is to improve sensitivity in detecting toxic effects of nanoplastics. In particular, the study focused on nano-sized carboxylated polystyrene (PS) nanoparticles. Two variability factors that could influence biological effects of nanoplastics, the particle size and the age of the organisms, were considered. Results demonstrated that the application of the proposed integrated approach allowed the detection of early subtle effects such as a significant impact on the heart rate and behavior of Daphnia magna under short-term exposure to PS carboxylated nanoparticles. In particular, a stimulation of heart rate was observed for both neonates and adults either for 40 nm or 200 nm particles after 48 h exposure, presumably attributable to an interference of carboxylated PS NPs with adrenergic-type receptors. Behavioral alterations were detectable for 40 nm particles but not for 200 nm ones consisting of a decrease in velocity and alterations of trajectories. Obtained results demonstrated the suitability of the proposed smartphone platform for friendly and real-time integration of behavioral analysis with physiological outcome measurements during acute exposure of Daphnia magna to nano-sized carboxylated PS NPs, expanding the sensitivity of the traditional acute toxicity tests. It offers a novel, cost-effective, and field-applicable method for environmental monitoring of nanoparticle toxicity and impact.

Bacterial cellulose (BC) hydrogel is a promising skin wound healing biomaterial due to its unique properties, including a moist environment that facilitates tissue healing. To enhance its antimicrobial efficacy, BC dressings were loaded with penicillin and streptomycin. FT-IR analysis confirmed successful drug binding, while SEM revealed a nanofibrous and porous hydrogel structure. In vitro studies using 3T3 mouse fibroblasts demonstrated biocompatibility, and scratch wound assays achieved complete closure across all tested concentrations. Antibacterial activity, assessed via agar diffusion against Pseudomonas aeruginosa and Staphylococcus aureus, showed a concentration-dependent increase in inhibition zones, highlighting the potential of BC-Pen/Strep hydrogels as effective antimicrobial wound dressings.

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate immunity. During the early stages of the immune response, hemocytes actively migrate to the site of infection. This inherent motility is a fundamental characteristic of these cells. It represents a key cellular function that integrates multiple processes, such as cell adhesion, cell signaling, cytoskeletal dynamics, and changes in cell volume. Therefore, alterations in cell motility following exposure to drugs or pollutants can serve as a useful toxicological endpoint. Despite the fundamental role of cell motility in cellular physiology, it has been poorly investigated from a toxicological perspective. This work proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on evaluating the hemocyte motility of Mytilus galloprovincialis. We developed a cell motility assay on hemocytes adhering to the bottom of a 96-well polystyrene microplate. Following exposure to increasing concentrations of drugs, cell trajectories, and velocities were quantified by cell tracking under time-lapse microscopy, allowing us to measure the effects on hemocyte motility. Due to the ease of hemocyte collection from the animals in a relatively non-invasive manner, the proposed method offers an alternative test for screening the effects and mechanisms of action of pollutants and drugs. It aligns with the 3Rs (Replacement, Reduction, and Refinement) criteria, addressing ethical concerns and contributing to the reduction of vertebrate in vivo animal testing.

This work contributes to fill one of the gaps regarding nanoplastic interactions with biological systems by producing polyethylene terephthalate (PET) model nanoplastics, similar to those found in the marine environment, by means of a fast top-down approach based on mechanical fragmentation. Their size distribution and morphology were characterized by laser diffraction and atomic force microscopy (AFM). Their autofluorescence was studied by spectrofluorimetry and fluorescence imaging, being a key property for the evaluation of their interaction with biota. The emission spectra of label-free nanoplastics were comparable with those of PET nanoplastics labeled with Nile red. Finally, the suitability of label-free nanoplastics for biological studies was assessed by in vitro exposure with Mytilus galloprovincialis hemolymphatic cells in a time interval up to 6 h. The nanoplastic internalization into these cells, known to be provided with phagocytic activity, was assessed by fluorescence microscopy. The obtained results underlined that the autofluorescence of the model PET nanoplastics produced in the laboratory was adequate for biological studies having the potential to overcome the disadvantages commonly associated with several fluorescent dyes, such as the tendency to also stain other organic materials different from plastics, to form aggregates due to intermolecular interactions at high concentrations with a consequent decrease in fluorescence intensity, and to dye desorption from nanoparticles. The results of the autofluorescence study provide an innovative approach for plastic risk assessment.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics. The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

Targeted therapies for cutaneous melanoma, such as those based on specific BRAF inhibitors, have improved the treatment and enhanced the survival rate of patients who harbor the V600E point mutation in the BRAF gene. However, tissue biopsies to characterize BRAF mutation status are prone to sampling bias, due to the intrinsic heterogeneity of a tumor mass. In contrast, blood biopsies, which analyze circulating tumor DNA (ctDNA), offer the most complete and sensitive characterization of the mutation status of a tumor, provide early and more accurate diagnosis, but they require instrumental and costly molecular tests. Therefore, the development of low-cost but highly sensitive tests for the non-invasive identification of BRAF^V600E mutation in ctDNA would be of great clinical utility as a routine screening for the early identification of responsive patients and the follow-up of targeted therapy's response. The present work developed a naked-eye, inexpensive, yet very specific colorimetric assay, whose sensitivity is suitable for the detection of BRAF^V600E rare mutation in ctDNA. Such test potentially may detect at an early stage the mutation in the tumor mass, when the first mutated cells appear in the blood, by using minimal instrumentation and thus enabling its widespread implementation in the clinics, even in local, minimally equipped laboratories. Indeed, the test detects 0.5% of BRAF^V600E in an excess of BRAF^WT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is thus clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients.

A method for the aqueous synthesis of stable and biocompatible citrate-coated palladium nanoparticles (PdNPs) in the size range comparable to natural enzymes (4–8 nm) has been developed. The toxicological profile of PdNPs was assessed by different assays on several cell lines demonstrating their safety in vitro also at high particle concentrations. To elucidate their cellular fate upon uptake, the localization of PdNPs was analyzed by Transmission Electron Microscopy (TEM). Moreover, crucial information about their intracellular stability and oxidation state was obtained by Sputtering-Enabled Intracellular X-ray Photoelectron Spectroscopy (SEI-XPS). TEM/XPS results showed significant stability of PdNPs in the cellular environment, an important feature for their biocompatibility and potential for biomedical applications. On the catalytic side, these PdNPs exhibited strong and broad antioxidant activities, being able to mimic the three main antioxidant cellular enzymes, i.e., peroxidase, catalase, and superoxide dismutase. Remarkably, using an experimental model of a human oxidative stress-related disease, we demonstrated the effectiveness of PdNPs as antioxidant nanozymes within the cellular environment, showing that they are able to completely re-establish the physiological Reactive Oxygen Species (ROS) levels in highly compromised intracellular redox conditions.