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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Bayesian inference has become a standard method of analysis in many fields of science. Students and researchers in experimental psychology and cognitive science, however, have failed to take full advantage of the new and exciting possibilities that the Bayesian approach affords. Ideal for teaching and self study, this book demonstrates how to do Bayesian modeling. Short, to-the-point chapters offer examples, exercises, and computer code (using WinBUGS or JAGS, and supported by Matlab and R), with additional support available online. No advance knowledge of statistics is required and, from the very start, readers are encouraged to apply and adjust Bayesian analyses by themselves. The book contains a series of chapters on parameter estimation and model selection, followed by detailed case studies from cognitive science. After working through this book, readers should be able to build their own Bayesian models, apply the models to their own data, and draw their own conclusions.

A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of ␣-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated ␣-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of ␣-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated ␣-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.A number of neurodegenerative diseases, including Parkinson disease (PD), 4 dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are defined histologically by the presence of Lewy bodies (LBs), intracellular protein aggregates that have a range of morphologies, from cytoplasmic spheres to neuritic threads also referred to as Lewy neurites (LNs). A number of proteins have been identified in LBs largely by immunohistochemical staining of brain, although the two most common are ubiquitin and ␣-synuclein (1-4). The invariable presence of ␣-synuclein in LBs suggests that it plays a key role in the etiology of such diseases ("synucleinopathies"). Point mutations in the synuclein gene as well as multiplication of the gene in familial cases of PD lead to autosomally dominant familial forms of PD (5-9). As in sporadic PD, LBs are also found in the brains of individuals with familial PD suggesting that clues about the pathogenic role of synuclein lie within the LB.Because ␣-synuclein is a relatively abundant neuronal protein, and LBs are found in diseased brain, we hypothesized that the formation of the abnormal LB structures results from specific modifications to this protein. We therefore analyzed the specific forms of ␣-synuclein that are found in LBs is...

Cerebral deposition of the beta-amyloid peptide (A beta) is an invariant feature of Alzheimer's disease. Since the original isolation and characterization of A beta (ref. 1) and the subsequent cloning of its precursor protein, no direct evidence for the actual production of discrete A beta has been reported. Here we investigate whether A beta is present in human biological fluids using antibodies specific for an epitope within A beta that spans the site of normal constitutive cleavage. These antibodies were used to construct a sandwich-type enzyme-linked immunosorbent assay that detects A beta in cerebrospinal fluid, plasma and conditioned medium of human mixed-brain cells grown in vitro (see also ref. 14). By affinity chromatography, we have purified and sequenced A beta and a novel A beta fragment from human cerebrospinal fluid and conditioned medium of human mixed-brain cell cultures. These findings demonstrate that A beta is produced and released both in vivo and in vitro. These observations offer new opportunities for developing diagnostic tests for Alzheimer's disease and therapeutic strategies aimed at reducing the cerebral deposition of A beta.

The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

No abstract

The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A beta 42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid beta-protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, alpha- and beta-secretase activity and A beta production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A beta 42 secretion in all mutant clones. Likewise, mutant but not wildtype PS1 transgenic mice showed significant overproduction of A beta 42 in the brain, and this effect was detectable as early as 2-4 months of age. Different PS mutations had differential effects on A beta generation. The extent of A beta 42 increase did not correlate with presenilin expression levels. Our data demonstrate that the presenilin mutations cause a dominant gain of function and may induce AD by enhancing A beta 42 production, thus promoting cerebral beta-amyloidosis.