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.

[6909][6910][6911][6912][6913][6914][6915][6916] 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.The hepatitis B virus (HBV) encodes a gene that produces a 154-amino-acid polypeptide, X. X has been shown to be essential for viral replication in animal hosts (7, 69), and it has also been implicated in the development of HBV-mediated carcinogenesis since X is able to induce liver tumors in certain transgenic mice strains (26,29,52). Although the exact role of X in the life cycle of the virus or in carcinogenesis is not known, it has been well established that X is a potent transactivator of a diverse number of viral and cellular promoters. X has been shown to activate RNA polymerase III (pol III)-dependent promoters (3, 58) and RNA pol II-dependent promoters which contain recognition sequences for AP-1, AP-2, ATF/CREB, c/EBP, NF-B, serum response factor, and a variety of acidic activator proteins (for reviews, see 43 and 68).There are at least two distinct mechanisms by which X appears to activate gene expression. For certain promoters, X may interact directly with the transcriptional machinery to augment RNA synthesis. Although X is not able to bind to DNA directly, it has been shown to bind to the CREB/ATF family of transcription factors and to enhance the affinity of the factor for its DNA recognition sequence (66) or alter its specificity (37). In addition, X has been shown to interact with the RPB5 subunit of the RNA polymerases (8, 33), the tumor suppressor p53 (15, 59), transcription factor IIB (TFIIB) (23,33), TFIIH (23,41), and the TATA-binding prot...

The hepatitis B virus X gene product transactivates a variety of cellular and viral genes. The mechanism for X induction of RNA polymerase (pol) III genes was investigated. By using Drosophila S-2 cells stably transformed with the X gene, the transient expression of a tRNA gene is enhanced. Comparing the transcriptional activities of extracts derived from these cells, all three types of RNA pol III promoters are stimulated by X. Interestingly, both S-2 and rat 1A cells stably transformed with the X gene produce increased cellular levels of the TATA-binding protein (TBP). By using various kinase inhibitors, it was found that the X-mediated increases in both transcription and TBP are dependent upon protein kinase C activation. Since TBP is a subunit of TFIIIB, the activity of this component fractionated from extracts derived from control and X-transformed cells was analyzed. These studies reveal that TFIIIB activity is substantially more limiting in control cells and that TFIIIB isolated from X-transformed cells has increased activity in reconstitution assays compared with TFIIIB isolated from control cells. Conversely, comparison of TFIIIC from control and X-transformed cell extracts revealed that there is relatively little change in its ability either to reconstitute transcription or to bind to DNA and that there is no change in the catalytic activity of RNA pol III. Studies were performed to determine whether directly increasing cellular TBP alone could enhance RNA pol III gene transcription. Transient expression of a TBP cDNA in rat 1A cells was capable of stimulating transcription activity from the resultant extracts in vitro. Together, these results demonstrate that one mechanism by which X mediates transactivation of RNA pol III genes is by increasing limiting TBP via the activation of cellular signaling pathways. The discovery that X increases cellular TBP, the universal transcription factor, provides a novel mechanism for the function of a viral transactivator protein and may explain the ability of X to produce such large and diverse effects on cellular gene expression.The hepatitis B virus (HBV) encodes four genes, one of which produces a 154-amino-acid polypeptide, X, which is known to transactivate a large variety of cellular and viral RNA polymerase (pol) II promoters as well as an RNA pol III promoter (51). There is evidence that X may play a significant role in the development of hepatocellular carcinoma, which occurs with high incidence following chronic infection by HBV (35). Hence, the mechanism of action of X has been the subject of intense study. The mechanism for X transactivation is not yet clear; however, several distinct models have been proposed. X does not bind to DNA directly, but several studies have provided evidence that X may interact with specific DNAbinding proteins to activate transcription. X has been shown to stimulate transcription when targeted to specific DNA-binding sites by fusion of X with heterologous DNA-binding domains (39,45). Other studies have demonstrated direct pro...

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