Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2017 KEY WORDS Review Autophagy and AgingThe Importance of Maintaining "Clean" Cells ABSTRACTA decrease in the turnover of cellular components and the intracellular accumulation of altered macromolecules and organelles are features common to all aged cells. Diminished autophagic activity plays a major role in these age-related manifestations. In this work we review the molecular defects responsible for the malfunctioning of two forms of autophagy, macroautophagy and chaperone-mediated autophagy, in old mammals, and highlight general and cell-type specific consequences of dysfunction of the autophagic system with age. Dietary caloric restriction and antilipolytic agents have been proven to efficiently stimulate autophagy in old rodents. These and other possible experimental restorative efforts are discussed.

In an effort to better understand the phenomenon of lipotoxicity in human ␤-cells, we evaluated the effects of 48-h preculture with 1.0 or 2.0 mmol/l free fatty acid (FFA) (2:1 oleate to palmitate) on the function and survival of isolated human islets and investigated some of the possible mechanisms. Compared with control islets, triglyceride content was significantly increased and insulin content and glucose-stimulated insulin release were significantly reduced in islets precultured with increased FFA concentrations. These changes were accompanied by a significant reduction of glucose utilization and oxidation. By cell death detection techniques, it was observed that exposure to FFAs induced a significant increase of the amount of dead cells. Electron microscopy showed the involvement of ␤-cells, with morphological appearance compatible with the presence of apoptotic phenomena. FFA-induced islet cell death was blocked by inhibition of upstream caspases and partially prevented by inhibiton of ceramide synthesis or serine protease activity, whereas inhibition of nitric oxide synthesis had no effect. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Thus, prolonged exposure to FFAs has cytostatic and proapoptotic effects on human pancreatic ␤-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism, and the proapoptotic effects are mostly caspase mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by ␣-tocopherol. We show here that ␣-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, ␣-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by ␣-tocopherol. However, ␣-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-␥ (PPAR␥; troglitazone), indicating that this pathway is susceptible to ␣-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser 473 was increased by oxLDL, and ␣-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPAR␥ element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPAR␥ and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPAR␥ signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.In many cell types, oxidized low density lipoproteins (oxLDL) 2 modulate cellular processes such as apoptosis, adhesion, migration, gene expression, and the induction of signal transduction cascades (1). Exposure of monocytes to oxLDL may alter gene expression and signaling, making them more susceptible to the following pro-atherogenic stimuli. The migration of monocytes into the intima and the conversion of monocytes/macrophages into foam cells represent initial steps in atherosclerosis. Current strategies to prevent atherosclerosis are aimed either at lowering the cholesterol load of lipoproteins or at reducing oxidative stress.Vitamin E is a redox-active natural compound that can act, depending on the conditions, as a pro-or antioxidant on low density lipoproteins (LDL) in vitro and in vivo (2-5). The major form of vitamin E in human plasma is ␣-tocopherol, and reduced plasma levels of ␣-tocopherol, such as in vitamin E-deficient mice, increase the incidence of atherosclerosis (6). Animal and cell culture studies strongly suggest that vitamin E can prevent atherosclerosis; however, the anti-atherogenic effects in clinical trials are still controversial (7-10). ␣-Tocopherol in lipoproteins (mainly LDL) and also in the subendothelial space has been assumed to play a central role in reducing atherosclerosis by preventing lipid peroxidation and consequent lesion development. Nevertheless, since many compounds exist that can interfere with the oxidation of LDL without being equally effective, alternative modes of action have bee...

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Aging denotes a postmaturational deterioration of cells and organisms with the passage of time, an increased vulnerability to challenges and prevalence of age-associated diseases, and a decreased ability to survive. Causes of this deterioration may be found in an enhanced production of reactive oxygen species (ROS) and oxidative damage and incomplete "housekeeping." Caloric restriction is the most robust anti-aging intervention known so far. Similar beneficial effects on median and maximum life span were obtained by feeding animals a 40%-reduced diet or by every-other-day ad libitum feeding. In both instances, animals are forced to spend a great part of their time in a state of fasting and activated autophagy. Autophagy is a highly conserved process in eukaryotes, in which the cytoplasm, including excess or aberrant organelles, is sequestered into double-membrane vesicles and delivered to the lysosome/vacuole, for breakdown and eventual recycling of the resulting macromolecules. This process has an essential role in adaptation to fasting and changing environmental conditions, cellular remodeling during development, and accumulation of altered ROS-hypergenerating organelles in older cells. Several pieces of evidence show that autophagy is involved in aging and is an essential part of the anti-aging mechanism of caloric restriction. As an application, intensification of autophagy by the administration of an antilipolytic drug rescued older cells from accumulation of altered mtDNA in less than 6 hours. It is concluded that the pharmacologic intensification of autophagy (PISA treatment) has anti-aging effects and might prove to be a big step toward retardation of aging and prevention of age-associated diseases in humans.

Fusarium mycotoxins are a relevant problem in the cereal supply chain at a worldwide level, with wheat, maize and barley being the main contaminated crops. Mould growth can happen in the pre-harvest phase and also during transport and storage due to ineffective drying conditions. Among Fusarium toxins, deoxynivalenol (DON) is considered the most important contaminant in wheat due to its widespread occurrence. In the last years the European Food Safety Authority (EFSA) and the European Commission have frequently expressed opinions on Fusarium toxins, setting limits, regulations and guidelines in order to reduce their levels in raw materials and food commodities. In particular, European legislation (Reg. 1881/2006) sets the maximum limit for DON in flour and bread as 750 and 500 microg kg(-1) respectively. Relatively few studies have taken into account the loss of trichothecenes during processing, focusing on how processing factors may influence their degradation. In particular, the description of DON behaviour during bread-making is very difficult, since complex physico-chemical modifications occur during the transformation of the raw ingredients into the final product. In the present study, we studied how DON concentration may be influenced by modifying bread-making parameters, with a special emphasis on the fermentation and baking stages, starting from a naturally contaminated flour at both pilot and industrial scales. Exploiting the power of a Design of Experiments (DoE) approach to consider the great complexity of the studied system, the obtained model shows satisfying goodness-of-fit and prediction, suggesting that the baking step (time/temperature ranges) is crucial for minimizing native DON level in bread.