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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field

Long-lived organisms tend to be more resistant to various forms of environmental stress. An example is the Drosophila longevity mutant, methuselah, which has enhanced resistance to heat, oxidants, and starvation. To identify genes regulated by these three stresses, we made a cDNA library for each by subtraction of ''unstressed'' from ''stressed'' cDNA and used DNA hybridization to identify genes that are regulated by all three. This screen indeed identified 13 genes, some already known to be involved in longevity, plus candidate genes. Two of these, hsp26 and hsp27, were chosen to test for their effects on lifespan by generating transgenic lines and by using the upstream activating sequence͞GAL4 system. Overexpression of either hsp26 or hsp27 extended the mean lifespan by 30%, and the flies also displayed increased stress resistance. The results demonstrate that multiple-stress screening can be used to identify new longevity genes.aging ͉ heat-shock proteins hsp26 and hsp27 ͉ chaperones ͉ paraquat ͉ starvation G enetic or environmental manipulations to extend lifespan in various organisms have been found to correlate with increases in resistance to environmental stress (1, 2). Drosophila selected for delay in age of reproduction have increased longevity and higher resistance to many stresses, including desiccation, heat, starvation, and oxidants (3, 4), and the long-lived Drosophila methuselah and ecdysone receptor (EcR) mutants show enhanced resistance to paraquat, starvation, and heat (5, 6). The long-lived Caenorhabditis elegans mutants age-1 and daf-2 have higher resistance to oxidative stress (7), ultraviolet light (8), and heat (9, 10). Skin fibroblasts taken from long-lived p66 shc knockout mice show increased resistance to oxidative stress and UV light (11). Skin fibroblasts from mammals with varying lifespan show a positive correlation between lifespan and resistance to a variety of stressors (12). Hormesis, the beneficial effect of exposure to sublethal stress, can extend longevity in C. elegans (13) and in Drosophila (14).The correlation between stress resistance and lifespan has prompted efforts to screen for lifespan extension mutants via increased stress resistance. Several such screens have been successful, including selection for paraquat resistance in Drosophila (15) and for increased thermotolerance in C. elegans (16,17). A screen for resistance to both heat and paraquat in yeast identified mutations in adenylate cyclase and Akt͞PKB that extended stationary-phase survival 3-fold (18).The link between stress resistance and lifespan extension is further strengthened by findings that manipulation of stressresponsive genes can extend lifespan. For example, Drosophila lifespan is increased by overexpression of the antioxidant Cu-Zn superoxide dismutase (SOD) (19)(20)(21) or by overexpression of the heat-shock protein (HSP) gene hsp70 (22). In the C. elegans mutant age-1, up-regulation of Hsp16 extends lifespan (23), and heat-shock factor 1 promotes longevity as well as resistance to heat and oxidati...

Dietary restriction extends lifespan in a variety of organisms, but the key nutritional components driving this process and how they interact remain uncertain. In Drosophila, while a substantial body of research suggests that protein is the major dietary component affecting longevity, recent studies claim that carbohydrates also play a central role. To clarify how nutritional factors influence longevity, nutrient consumption and lifespan were measured on a series of diets with varying yeast and sugar content. We show that optimal lifespan requires both high carbohydrate and low protein consumption, but neither nutrient by itself entirely predicts lifespan. Increased dietary carbohydrate or protein concentration does not always result in reduced feeding—the regulation of food consumption is best described by a constant daily caloric intake target. Moreover, due to differences in food intake, increased concentration of a nutrient within the diet does not necessarily result in increased consumption of that particular nutrient. Our results shed light on the issue of dietary effects on lifespan and highlight the need for accurate measures of nutrient intake in dietary manipulation studies.

Food and other environmental factors affect gene expression and behaviour of animals. Differences in bacterial food affect the behaviour and longevity of Caenorhabditis elegans. However, no research has been carried out to investigate whether bacteria could utilize endogenous RNAs to affect C. elegans physiology. Here we show that two Escherichia coli endogenous noncoding RNAs, OxyS and DsrA, impact on the physiology of C. elegans. OxyS downregulates che-2, leading to impairment in C. elegans chemosensory behaviour and DsrA suppresses diacylglycerol lipase gene F42G9.6, leading to a decrease in longevity. We also examine some genes in the C. elegans RNA interference pathway for their possible involvement in the effects of OxyS and DsrA. Other bacteria, such as Bacillus mycoides, may also utilize its noncoding RNAs to interfere with gene expression in C. elegans. Our results demonstrate that E. coli noncoding RNAs can regulate gene expression and physiological conditions of C. elegans and indicate that noncoding RNAs might have interspecies ecological roles.

Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.