In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Ecotoxicity and genotoxicity of polystyrene microplastics on higher plant Vicia faba.

Cerium oxide nanoparticles (CeNPs) exhibit antioxidant properties both in vitro and in vivo This is due to the self-regeneration of their surface, which is based on redox-cycling between 3+ and 4+ states for cerium, in response to their immediate environment Additionally, oxygen vacancies in the lattice structure allow for alternating between CeO2 and CeO2−x during redox reactions Research to identify and characterize the biomedical applications of CeNPs has been heavily focused on investigating their use in treating diseases that are characterized by higher levels of reactive oxygen species (ROS) Although the bio-mimetic activities of CeNPs have been extensively studied in vitro, in vivo interactions and associated protein corona formation are not well understood This review describes: (1) the methods of synthesis for CeNPs, including the recent green synthesis methods that offer enhanced biocompatibility and a need for establishing a reference CeNP material for consistency across studies; (2) their enzyme-mimetic activities, with a focus on their antioxidant activities; and, (3) recent experimental evidence that demonstrates their ROS scavenging abilities and their potential use in personalized medicine

https://www.mdpi.com/2076-3921/7/8/97/pdf/1

Cerium Oxide Nanoparticles: A Brief Review of Their Synthesis Methods and Biomedical Applications.

Engineered nanomaterials for plant growth and development: A perspective analysis.

Previously, catalytic cerium oxide nanoparticles (CNPs, nanoceria, CeO2-x NPs) have been widely utilized for chemical mechanical planarization in the semiconductor industry and for reducing harmful emissions and improving fuel combustion efficiency in the automobile industry. Researchers are now harnessing the catalytic repertoire of CNPs to develop potential new treatment modalities for both oxidative- and nitrosative-stress induced disorders and diseases. In order to reach the point where our experimental understanding of the antioxidant activity of CNPs can be translated into useful therapeutics in the clinic, it is necessary to evaluate the most current evidence that supports CNP antioxidant activity in biological systems. Accordingly, the aims of this review are three-fold: (1) To describe the putative reaction mechanisms and physicochemical surface properties that enable CNPs to both scavenge reactive oxygen species (ROS) and to act as antioxidant enzyme-like mimetics in solution; (2) To provide an overview, with commentary, regarding the most robust design and synthesis pathways for preparing CNPs with catalytic antioxidant activity; (3) To provide the reader with the most up-to-date in vitro and in vivo experimental evidence supporting the ROS-scavenging potential of CNPs in biology and medicine.

Antioxidant Cerium Oxide Nanoparticles in Biology and Medicine

Nanoremediation of soil, ground and surface water using nanoscale zerovalent iron particles (nZVI) has facilitated their direct environmental exposure posing ecotoxicological concerns. Numerous studies elucidate their phytotoxicity in terms of growth and their fate within the plant system. However, their potential genotoxicity and cytotoxicity mechanisms are not known in plants. This study encompasses the physico-chemical characterisation of two forms of nZVI (nZVI-1 and nZVI-2) with different surface chemistries and their influence on uptake, root morphology, DNA damage, oxidative stress and cell death in Allium cepa roots after 24 h. To our knowledge, this is the first report on the cyto-genotoxicity of nZVI in plants. The adsorption of nZVI on root surfaces caused root tip, epidermal and root hair damage as assessed by Scanning Electron Microscopy. nZVI-1, due to its colloidal destabilisation (low zeta potential, conductivity and high polydispersity index), smaller size and high uptake imparted enhanced DNA damage, chromosome/nuclear aberrations (CAs/NAs) and micronuclei formation compared to nZVI-2. Although nZVI-2 exhibited high zeta potential and conductivity, its higher dissolution and substantial uptake induced genotoxicity. nZVI incited the generation of reactive oxygen species (ROS) (hydrogen peroxide, superoxide and hydroxyl radicals) leading to membrane lipid peroxidation, electrolyte leakage and mitochondrial depolarisation. The inactivation of catalase and insignificant glutathione levels marked the onset of oxidative stress. Increased superoxide dismutase and guaiacol peroxidase enzyme activities, and proline content indicated the activation of antioxidant defence machinery to alleviate ROS. Moreover, ROS-mediated apoptotic and necrotic cell death occurred in both nZVI-1 and nZVI-2-treated roots. Our results open up further possibilities in the environmental safety appraisal of bare and modified nZVI in correlation with their physico-chemical characters.

In planta genotoxicity of nZVI: influence of colloidal stability on uptake, DNA damage, oxidative stress and cell death.

Nanoparticles (NPs) are an emerging environmental threat. However, studies of NPs in different environmental components are limited. In this review, we discuss studies that have evaluated the genotoxicity of NPs in higher plants. Among the 29 studies reviewed, silver NPs were most studied (n = 7 articles), with fewer studies reporting the genotoxicity of carbon nanotubes (n = 3), titanium dioxide NPs (n = 4), and zinc oxide NPs (n = 3). Most of the genotoxicity studies were performed in the model plant systems Allium sp (n = 22), Nicotiana sp (n = 4) and Vicia sp (n = 4) using chromosome aberration (n = 22), micronucleus (n = 15) and comet assays (n = 14). Genotoxicity was observed in most of the studies; however, many studies did consider key determinants of NP toxicity such as particle characterization, dissolution, and uptake. From this review, we propose a set of guidelines that should be considered when reporting results of NP toxicity in plants.

Genotoxicity of engineered nanoparticles in higher plants.

Genotoxicity and biocompatibility of superparamagnetic iron oxide nanoparticles: Influence of surface modification on biodistribution, retention, DNA damage and oxidative stress.

In the present study, we examined the effects of aluminium oxide nanoparticles (Al2O3 NPs) and bulk aluminium oxide (Al2O3) on Allium cepa in terms of genotoxicity and oxidative stress responses under in planta conditions. The concentrations ranged from 1.25 to 5 µM for NP and its bulk form. Results from this investigation indicated steady rise in chromosome aberrations, micronuclei and DNA strand breaks with increase in concentration of the NPs, effects of Al2O3 NPs being significantly higher than those treated with bulk Al2O3. The divisional frequency was affected by the NP treatment. Oxidative damage such as lipid peroxidation and cell death to Allium under Al2O3 NPs treatments was greater than that observed in bulk Al2O3. A significant increase in activity of guiacol peroxidase was observed in accordance with the depletion in catalase activity in both Al2O3 NPs and bulk Al2O3 treated plants as compared to control. In conclusion, the present study indicated that Al2O3 NPs treatment exerted higher genotoxicity and oxidative stress responses on A. cepa than bulk Al2O3 under in planta conditions.

Evaluation of genotoxicity and oxidative stress of aluminium oxide nanoparticles and its bulk form in Allium cepa

The effect of cerium oxide nanoparticle (CeNP) in plants has elicited substantial controversy. While some investigators have reported that CeNP possesses antioxidant properties, others observed CeNP to induce reactive oxygen species (ROS). In spite of considerable research carried out on the effects of CeNP in metazoans, fundamental studies that can unveil its intracellular consequences linking ROS production, autophagy and DNA damage are lacking in plants. To elucidate the impact of CeNP within plant cells, tobacco BY-2 cells were treated with 10, 50 and 250 µg ml-1 CeNP (Ce10, Ce50 and Ce250), for 24 h. Results demonstrated concentration-dependent accumulation of Ca2+ and ROS at all CeNP treatment sets. However, significant DNA damage and alteration in antioxidant defence systems were noted prominently at Ce50 and Ce250. Moreover, Ce50 and Ce250 induced DNA damage, analysed by comet assay and DNA diffusion experiments, complied with the concomitant increase in ROS. Furthermore, to evaluate the antioxidant property of CeNP, treated cells were washed after 24 h (to minimise CeNP interference) and challenged with H2O2 for 3 h. Ce10 did not induce genotoxicity and H2O2 exposure to Ce10-treated cells showed lesser DNA breakage than cells treated with H2O2 only. Interestingly, Ce10 provided better protection over N-acetyl-L-cysteine against exogenous H2O2 in BY-2 cells. CeNP exposure to transgenic BY-2 cells expressing GFP-Atg8 fusion protein exhibited formation of autophagosomes at Ce10. Application of vacuolar protease inhibitor E-64c and fluorescent basic dye acridine orange, further demonstrated accumulation of particulate matters in the vacuole and occurrence of acidic compartments, the autophagolysosomes, respectively. BY-2 cells co-treated with CeNP and autophagy inhibitor 3-methyladenine exhibited increased DNA damage in Ce10 and cell death at all assessed treatment sets. Thus, current results substantiate an alternative autophagy-mediated, antioxidant and geno-protective role of CeNP, which will aid in deciphering novel phenomena of plant-nanoparticle interaction at cellular level.

Ilika Ghosh

Papers

In planta genotoxicity of nZVI: influence of colloidal stability on uptake, DNA damage, oxidative stress and cell death.

Genotoxicity of engineered nanoparticles in higher plants.

Genotoxicity and biocompatibility of superparamagnetic iron oxide nanoparticles: Influence of surface modification on biodistribution, retention, DNA damage and oxidative stress.

Evaluation of genotoxicity and oxidative stress of aluminium oxide nanoparticles and its bulk form in Allium cepa

Role of cerium oxide nanoparticle-induced autophagy as a safeguard to exogenous H2O2-mediated DNA damage in tobacco BY-2 cells.