Emilia Maellaro

Bio: Emilia Maellaro is an academic researcher from University of Siena. The author has contributed to research in topics: Glutathione & Lipid peroxidation. The author has an hindex of 33, co-authored 62 publications receiving 8056 citations. Previous affiliations of Emilia Maellaro include Karolinska Institutet.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Lipid peroxidation and cellular damage in extrahepatic tissues of bromobenzene-intoxicated mice

Abstract: The mechanisms of bromobenzene toxicity in extrahepatic tissues of mice were studied. Kidney, lung, heart and brain were examined. As observed in this as well as in a previous report for the liver, bromobenzene intoxication caused a progressive decrease in the glutathione content of all the tissues examined. Cellular damage (as assessed by both biochemical determinations and histologic observations) appeared after 6 hours in the case of the kidney and the heart and after 15 hours in the case of the lung. Lipid peroxidation (as assessed by the tissue content of malonic dialdehyde, a parameter correlating with both the diene conjugation absorption and the amount of carbonyl functions in cellular phospholipids) was found to occur at the same times at which cellular damage was observed or even before. As in the case of bromobenzene-induced liver injury, when the individual values for cell damage obtained at 15-20 hours were plotted against the corresponding glutathione contents, a severe cellular damage was generally observed when the glutathione levels reached a threshold value (3.0-0.5 nmol/mg protein). Such a glutathione threshold was also observed for the onset of lipid peroxidation. Glutathione depletion and lipid peroxidation are therefore general phenomena occurring not only in the liver but in all the tissues as a consequence of bromobenzene poisoning. The possibility that lipid peroxidation is the cause of bromobenzene-induced damage to liver and extrahepatic tissues is discussed.

Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway of thymocyte apoptosis

Abstract: Oxidative stress has recently been suggested to be a mediator of apoptotic cell death [Buttke and Sandstrom (1994) Immunology Today 15, 7-10], although evidence that this phenomenon is a widespread component of apoptosis is lacking. When rat thymocytes were exposed to the glucocorticoid methylprednisolone (MPS), a progressive increase in intracellular peroxides and a decrease in glutathione (GSH) were observed to accompany the onset of apoptosis. Using Percoll density gradients to isolate subpopulations of thymocytes at different stages of apoptosis, the increase in peroxide content was found to be restricted to apoptotic cells, while a significant depletion of GSH and reduced protein thiol was detected in both pre-apoptotic and fully apoptotic cells. To investigate the biological significance of these redox changes, the free radical spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide (TMPO), and the related nitroxide-radical antioxidant 2,2,6,6-tetramethyl-1-piperidinyl-1-oxyl (TEMPO) were tested as inhibitors of thymocyte apoptosis. The cell shrinkage and DNA fragmentation induced by four different initiators of apoptosis were reduced by each compound. TEMPO inhibition of both etoposide- and MPS-induced thymocyte DNA fragmentation was also found to correlate with an increase in intracellular GSH, providing support for the proposal that its antioxidant properties were responsible for the observed protective activity. We conclude that some form of intracellular oxidation (here measured indirectly by changes in intracellular GSH and peroxide levels) is required during thymocyte apoptosis even when this process is initiated by an agent that does not exert a direct oxidant action.

Erythrocyte caspase-3 activation and oxidative imbalance in erythrocytes and in plasma of type 2 diabetic patients.

Abstract: An increased oxidative stress and a decreased life span of erythrocytes (RBCs) are reported in patients with diabetes. Aim of this study was to assess in RBCs from patients with type 2 diabetes whether downstream effector mechanisms of apoptosis, such as activation of caspase-3, is operative, and whether an iron-related oxidative imbalance, occurring inside RBCs and in plasma, could be involved in caspase-3 activation. In 26 patients with type 2 diabetes and in 12 healthy subjects, oxidative stress was evaluated by means of different markers; non-protein-bound iron, methemoglobin and glutathione were determined in RBCs, and non-protein-bound iron was also determined in plasma. Erythrocyte caspase-3 activation was evaluated by an immunosorbent enzyme assay. Arterial hypertension, demographic and standard biochemical data were also evaluated. The results show, for the first time, that type 2 diabetic RBCs put into motion caspase-3 activation, which is significantly higher than in control RBCs. Such an effector mechanism of “eryptosis” was positively correlated to blood glucose levels and to the increased plasma NPBI level. Caspase-3 activation was also positively correlated to occurrence of arterial hypertension. The results suggest that an extracellular oxidative milieu can be responsible for erythrocyte caspase-3 activation in patients with type 2 diabetes. In turn, caspase-3 activation can be envisaged as a novel mechanism which, by impairing the maintenance of erythrocyte shape and function, might contribute to the shortened life span of RBCs from patients with type 2 diabetes and to hemorheological disorders observed in these patients.

I and i

Abstract: There is, I think, something ethereal about i —the square root of minus one. I remember first hearing about it at school. It seemed an odd beast at that time—an intruder hovering on the edge of reality. Usually familiarity dulls this sense of the bizarre, but in the case of i it was the reverse: over the years the sense of its surreal nature intensified. It seemed that it was impossible to write mathematics that described the real world in …

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds

Abstract: The occurrence of malondialdehyde (MDA), a secondary end product of the oxidation of polyunsatu- rated fatty acids, is considered a useful index of general lipid peroxidation A common method for measuring MDA, referred to as the thiobarbituric acid-reactive- substances (TBARS) assay, is to react it with thiobar- bituric acid (TBA) and record the absorbance at 532 nm However, many plants contain interfering compounds that also absorb at 532 nm, leading to overestimation of MDA values Extracts of plant tissues including purple eggplant (Solanum melongena L) fruit, carrot (Daucus carota L) roots, and spinach (Spinacia oleracea L) leaves were assessed for the presence of MDA and other non-MDA compounds absorbing at 532 nm A method described herein corrects for these interferences by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA The reliability and eAciency of this spectrophotometric method was assessed by altering the relative ratios of exogenous MDA additions and/or extracts of red cabbage (Brassica oleracea L) leaves containing inter- fering compounds and then measuring MDA recovery Reliability was also validated through high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry techniques Results indicated that over 90% of exogenously added MDA could be recovered through the improved protocol If there were no corrections for interfering compounds, MDA equivalents were overestimated by up to 965% Interfering compounds were not detected in vegetables such as lettuce (Lactuca sativa L) and spinach which had low or negligible concentrations of anthocyanidin derivatives Comparisons between the TBARS method presented here and two currently accepted protocols indicated that the new modified method exhibits greater accuracy for quantifying TBA-MDA levels in tissues containing anthocyanins and/or other interfering com- pounds This modified protocol represents a facile and rapid method for assessment of lipid peroxidation in virtually all plant species that contain interfering com- pounds

Cisplatin: mode of cytotoxic action and molecular basis of resistance

Abstract: Cisplatin is one of the most potent antitumor agents known, displaying clinical activity against a wide variety of solid tumors. Its cytotoxic mode of action is mediated by its interaction with DNA to form DNA adducts, primarily intrastrand crosslink adducts, which activate several signal transduction pathways, including those involving ATR, p53, p73, and MAPK, and culminate in the activation of apoptosis. DNA damage-mediated apoptotic signals, however, can be attenuated, and the resistance that ensues is a major limitation of cisplatin-based chemotherapy. The mechanisms responsible for cisplatin resistance are several, and contribute to the multifactorial nature of the problem. Resistance mechanisms that limit the extent of DNA damage include reduced drug uptake, increased drug inactivation, and increased DNA adduct repair. Origins of these pharmacologic-based mechanisms, however, are at the molecular level. Mechanisms that inhibit propagation of the DNA damage signal to the apoptotic machinery include loss of damage recognition, overexpression of HER-2/neu, activation of the PI3-K/Akt (also known as PI3-K/PKB) pathway, loss of p53 function, overexpression of antiapoptotic bcl-2, and interference in caspase activation. The molecular signature defining the resistant phenotype varies between tumors, and the number of resistance mechanisms activated in response to selection pressures dictates the overall extent of cisplatin resistance.