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

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Intraductal papillary mucinous tumors (IPMTs) of the pancreas may be meaningfully construed as representing 2 clinically distinct subtypes: main duct tumors (MDT) and branch duct tumors (BDT).

Carotenoids are thought to be the precursors of terpenoid volatile compounds that contribute to flavor and aroma. One such volatile, b-ionone, is important to fragrance in many flowers, including petunia (Petunia hybrida). However, little is known about the factors regulating its synthesis in vivo. The petunia genome contains a gene encoding a 9,10(9#,10#) carotenoid cleavage dioxygenase, PhCCD1. The PhCCD1 is 94% identical to LeCCD1A, an enzyme responsible for formation of b-ionone in tomato (Lycopersicon esculentum; Simkin AJ, Schwartz SH, Auldridge M, Taylor MG, Plant J [in press]). Reduction of PhCCD1 transcript levels in transgenic plants led to a 58% to 76% decrease in b-ionone synthesis in the corollas of selected petunia lines, indicating a significant role for this enzyme in volatile synthesis. Quantitative reverse transcription-PCR analysis revealed that PhCCD1 is highly expressed in corollas and leaves, where it constitutes approximately 0.04% and 0.02% of total RNA, respectively. PhCCD1 is light-inducible and exhibits a circadian rhythm in both leaves and flowers. b-Ionone emission by flowers occurred principally during daylight hours, paralleling PhCCD1 expression in corollas. The results indicate that PhCCD1 activity and b-ionone emission are likely regulated at the level of transcript.Apocarotenoids are a class of compounds derived from oxidative cleavage of carotenoids that are important contributors to flavor and fragrance of foods (Walhberg and Eklund, 1998). Until recently, the derivation of many of these compounds from carotenoids was largely based on structural considerations and correlations between levels of substrates and products (Buttery et al., 1988). With more than 600 carotenoids identified to date, apocarotenoids constitute one of the largest classes of molecules in nature. Some of these apocarotenoids are essential and valuable constituents of color, flavor, and aroma (Winterhalter and Rouseff, 2002).Recently, a family of enzymes that could potentially generate many apocarotenoids has been described. This family, the carotenoid cleavage dioxygenases (CCDs), has been shown to cleave multiple carotenoids at specific double bonds within the substrate (Schwartz et al., 2001;Giuliano et al., 2003). One of the best-characterized apocarotenoids is the hormone abscisic acid (ABA). ABA is a C 15 compound derived from 11,12 cleavage of the epoxy-carotenoids 9-cisvioloaxanthin and 9-cis-neoxanthin by VP14 to produce xanthoxin Tan et al., 1997). VP14 is the founding member of this unique family of dioxygenases. In Arabidopsis (Arabidopsis thaliana), there are nine members of the CCD family, five of which are believed to be involved in ABA synthesis (Tan et al., 2003). One member of the family, AtCCD1 that is not involved in ABA synthesis, symmetrically cleaves the 9,10(9#,10#) double bonds of multiple carotenoid substrates in vitro. Homologues of this enzyme, which generates a C 14 dialdehyde and two C 13 products, have been identified in Phaseolus vulgaris (Schwartz et al., 2001), crocus (Cr...

The molecular mechanisms responsible for postpollination changes in floral scent emission were investigated in snapdragon cv Maryland True Pink and petunia cv Mitchell flowers using a volatile ester, methylbenzoate, one of the major scent compounds emitted by these flowers, as an example. In both species, a 70 to 75% pollination-induced decrease in methylbenzoate emission begins only after pollen tubes reach the ovary, a process that takes between 35 and 40 h in snapdragon and ‫ف‬ 32 h in petunia. This postpollination decrease in emission is not triggered by pollen deposition on the stigma. Petunia and snapdragon both synthesize methylbenzoate from benzoic acid and S -adenosyl-L -methionine (SAM); however, they use different mechanisms to downregulate its production after pollination. In petunia, expression of the gene responsible for methylbenzoate synthesis is suppressed by ethylene. In snapdragon, the decrease in methylbenzoate emission is the result of a decrease in both S -adenosyl-L -methionine:benzoic acid carboxyl methyltransferase (BAMT) activity and the ratio of SAM to S -adenosyl-L -homocysteine ("methylation index") after pollination, although the BAMT gene also is sensitive to ethylene.

Transcriptional activity of HIF-1 (hypoxia-inducible factor-1) has been reported to be up-regulated in solid tumors after ionizing radiation; however, the molecular mechanism underlying the response remains to be elucidated. In the present study, we performed a series of molecular imaging experiments using a HIF-1-dependent reporter gene, 5HREp-ODD-luc, and found an essential role of the Akt/mTOR pathway.

In many flowering plants, such as petunia (Petunia 3 hybrida), ethylene produced in floral organs after pollination elicits a series of physiological and biochemical events, ultimately leading to senescence of petals and successful fertilization. Here, we demonstrate, using transgenic ethylene insensitive (44568) and Mitchell Diploid petunias, that multiple components of emission of volatile organic compounds (VOCs) are regulated by ethylene. Expression of benzoic acid/salicylic acid carboxyl methyltransferase (PhBSMT1 and 2) mRNA is temporally and spatially down-regulated in floral organs in a manner consistent with current models for postpollination ethylene synthesis in petunia corollas. Emission of methylbenzoate and other VOCs after pollination and exogenous ethylene treatment parallels a reduction in PhBSMT1 and 2 mRNA levels. Under cyclic light conditions (day/night), PhBSMT mRNA levels are rhythmic and precede emission of methylbenzoate by approximately 6 h. When shifted into constant dark or light conditions, PhBSMT mRNA levels and subsequent methylbenzoate emission correspondingly decrease or increase to minimum or maximum levels observed during normal conditions, thus suggesting that light may be a more critical influence on cyclic emission of methylbenzoate than a circadian clock. Transgenic PhBSMT RNAi flowers with reduced PhBSMT mRNA levels show a 75% to 99% decrease in methylbenzoate emission, with minimal changes in other petunia VOCs. These results implicate PhBSMT1 and 2 as genes responsible for synthesis of methylbenzoate in petunia.Many flowers exhibit a colorful display of petals and emit a complex mixture of floral volatile organic compounds (VOCs) that are together attractive to both pollinators and humans. Regulation of floral volatiles corresponds to pollinator activity times and receptivity of the flower to a pollination event (Dudareva et al., 2000;Schiestl and Ayasse, 2001). In many flowers, physiological changes take place following pollination and fertilization including petal wilting and abscission, color changes, flower closure, fruit development, and seed development (for review, see O'Neill, 1997). The plant hormone ethylene has been shown to coordinate several of these postpollination processes in many different plant species (van Doorn, 1997).In many plants, ethylene is synthesized and perceived in a localized, specific, and reproducible manner after pollination, underscoring the importance of understanding the progression of events and role of ethylene during pollination and fertilization. Petunia (Petunia 3 hybrida) is an excellent model system for studying postpollination responses because ethylene synthesis has been well characterized (Hoekstra and Weges, 1986;Tang and Woodson, 1996;Jones et al., 2003) and components of the ethylene-signaling pathway have been investigated (Wilkinson et al., 1997;Shibuya et al., 2004). In petunia, ethylene synchronizes pollen tube growth (Holden et al., 2003) and petal wilting (Hoekstra and Weges, 1986;Gubrium et al., 2000). An initial bur...