| 2. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)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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| 3. |
- Travassos, Maria, et al.
(författare)
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Does Caffeine Consumption Modify Cerebrospinal Fluid Amyloid-β Levels in Patients with Alzheimer's Disease?
- 2015
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Ingår i: Journal of Alzheimer's disease : JAD. - 1875-8908. ; 47:4, s. 1069-78
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Tidskriftsartikel (refereegranskat)abstract
- Caffeine may be protective against Alzheimer's disease (AD) by modulating amyloid-β (Aβ) metabolic pathways. The present work aimed to study a possible association of caffeine consumption with the cerebrospinal fluid (CSF) biomarkers, particularly Aβ. The study included 88 patients with AD or mild cognitive impairment. The consumption of caffeine and theobromine was evaluated using a validated food questionnaire. Quantification of caffeine and main active metabolites was performed with liquid chromatography coupled to tandem mass spectrometry. The levels of A(1-42), total tau, and phosphorylated tau in the CSF were determined using sandwich ELISA methods and other Aβ species, Aβ(X-38), Aβ(X-40), and Aβ(X-42), with the MSD Aβ Triplex assay. The concentration of caffeine was 0.79±1.15 μg/mL in the CSF and 1.20±1.88 μg/mL in the plasma. No correlation was found between caffeine consumption and Aβ42 in the CSF. However, a significant positive correlation was found between the concentrations of theobromine, both in the CSF and in the plasma, with Aβ42 in the CSF. Theobromine in the CSF was positively correlated with the levels of other xanthines in the CSF, but not in the plasma, suggesting that it may be formed by central metabolic pathways. In conclusion, caffeine consumption does not modify the levels of CSF biomarkers, and does not require to be controlled for when measuring CSF biomarkers in a clinical setting. Since theobromine is associated with a favorable Aβ profile in the CSF, the possibility that it might have a protective role in AD should be further investigated.
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