| 1. |
- Klionsky, Daniel J., et al.
(författare)
-
Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
-
Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
-
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.
|
|
| 2. |
- McCall, Matthew B B, et al.
(författare)
-
Persistence of full-length caspase-12 and its relation to malaria in West and Central African populations
- 2010
-
Ingår i: European Cytokine Network. - 1148-5493 .- 1952-4005. ; 21:2, s. 77-83
-
Tidskriftsartikel (refereegranskat)abstract
- Background. The full-length (L-) variant of caspase-12 is believed to predispose to sepsis. It has been replaced in the genome of most human populations by the (S-) variant, which leads to premature termination of translation. Strikingly, the L-allele is still widely prevalent in African populations, presumably due to a counterbalancing selective force specific to this continent, for which malaria is a prime candidate.Methods. We investigated associations between caspase-12 genotype and malarial parameters in three West-African populations, in studies encompassing immunological, clinical and obstetric data. Results. The caspase-12 L-allele was found at frequencies of 11-34%. Plasmodium falciparum-stimulated mononuclear cells from S/L heterozygote donors produced stronger interferon-γ and interleukin-10 responses than S/S homozygotes (p = 0.011 and p = 0.023 in uninfected and infected donors respectively). Nevertheless, we found no association between caspase-12 genotype and either the presentation of severe malaria or individual clinical parameters in sick children. Amongst pregnant women, the caspase-12 genotype did not influence peripheral or placental malaria infection, or basic obstetric parameters. Interestingly, perinatal mortality was more frequent in children of both S/S and L/L than S/L mothers, independent of placental P. falciparum-infection.Conclusion. We find little clinical or epidemiological evidence that malaria has contributed to the persistence of functional caspase-12 in Africa, suggesting either that alternative selective forces are at work or that genetic drift underlies its current global distribution.
|
|
