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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- 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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- Milosch, N, et al.
(author)
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Holo-APP and G-protein-mediated signaling are required for sAPPa-induced activation of the Akt survival pathway
- 2014
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In: Cell Death and Disease. - : Springer Science and Business Media LLC. - 2041-4889. ; 5
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Journal article (peer-reviewed)abstract
- Accumulating evidence indicates that loss of physiologic amyloid precursor protein (APP) function leads to reduced neuronal plasticity, diminished synaptic signaling and enhanced susceptibility of neurons to cellular stress during brain aging. Here we investigated the neuroprotective function of the soluble APP ectodomain sAPPα (soluble APPα), which is generated by cleavage of APP by α-secretase along the non-amyloidogenic pathway. Recombinant sAPPα protected primary hippocampal neurons and SH-SY5Y neuroblastoma cells from cell death induced by trophic factor deprivation. We show that this protective effect is abrogated in neurons from APP-knockout animals and APP-depleted SH-SY5Y cells, but not in APP-like protein 1- and 2- (APLP1 and APLP2) depleted cells, indicating that expression of membrane-bound holo-APP is required for sAPPα-dependent neuroprotection. Trophic factor deprivation diminished the activity of the Akt survival pathway. Strikingly, both recombinant sAPPα and the APP-E1 domain were able to stimulate Akt activity in wild-type (wt) fibroblasts, SH-SY5Y cells and neurons, but failed to rescue in APP-deficient neurons or fibroblasts. The ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) inhibitor GI254023X exacerbated neuron death in organotypic (hippocampal) slice cultures of wt mice subjected to trophic factor and glucose deprivation. This cell death-enhancing effect of GI254023X could be completely rescued by applying exogenous sAPPα. Interestingly, sAPPα-dependent Akt induction was unaffected in neurons of APP-ΔCT15 mice that lack the C-terminal YENPTY motif of the APP intracellular region. In contrast, sAPPα-dependent rescue of Akt activation was completely abolished in APP mutant cells lacking the G-protein interaction motif located in the APP C-terminus and by blocking G-protein-dependent signaling with pertussis toxin. Collectively, our data provide new mechanistic insights into the physiologic role of APP in antagonizing neurotoxic stress: they suggest that cell surface APP mediates sAPPα-induced neuroprotection via G-protein-coupled activation of the Akt pathway.
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- Kapitza, R., et al.
(author)
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CheapBFT : Resource-efficient Byzantine fault tolerance
- 2012
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In: EuroSys'12 - Proceedings of the EuroSys 2012 Conference. - New York : Association for Computing Machinery (ACM). - 9781450312233 ; , s. 295-308
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Conference paper (peer-reviewed)abstract
- One of the main reasons why Byzantine fault-tolerant (BFT) systems are not widely used lies in their high resource consumption: 3f + 1 replicas are necessary to tolerate only f faults. Recent works have been able to reduce the minimum number of replicas to 2f + 1 by relying on a trusted subsystem that prevents a replica from making conflicting statements to other replicas without being detected. Nevertheless, having been designed with the focus on fault handling, these systems still employ a majority of replicas during normal-case operation for seemingly redundant work. Furthermore, the trusted subsystems available trade off performance for security; that is, they either achieve high throughput or they come with a small trusted computing base. This paper presents CheapBFT, a BFT system that, for the first time, tolerates that all but one of the replicas active in normal-case operation become faulty. CheapBFT runs a composite agreement protocol and exploits passive replication to save resources; in the absence of faults, it requires that only f + 1 replicas actively agree on client requests and execute them. In case of suspected faulty behavior, CheapBFT triggers a transition protocol that activates f extra passive replicas and brings all non-faulty replicas into a consistent state again. This approach, for example, allows the system to safely switch to another, more resilient agreement protocol. CheapBFT relies on an FPGA-based trusted subsystem for the authentication of protocol messages that provides high performance and comprises a small trusted computing base.
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