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- 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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| 2. |
- Reinius, Björn, et al.
(författare)
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Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse
- 2010
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 11:1, s. 614-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Sexual dimorphism in brain gene expression has been recognized in several animal species.However, the relevant regulatory mechanisms remain poorly understood. To investigatewhether sex-biased gene expression in mammalian brain is globally regulated or locallyregulated in diverse brain structures, and to study the genomic organisation of brain-expressedsex-biased genes, we performed a large scale gene expression analysis of distinct brainregions in adult male and female mice. Results: This study revealed spatial specificity in sex-biased transcription in the mouse brain, andidentified 173 sex-biased genes in the striatum; 19 in the neocortex; 12 in the hippocampusand 31 in the eye. Genes located on sex chromosomes were consistently over-represented inall brain regions. Analysis on a subset of genes with sex-bias in more than one tissue revealedY-encoded male-biased transcripts and X-encoded female-biased transcripts known to escapeX-inactivation. In addition, we identified novel coding and non-coding X-linked genes withfemale-biased expression in multiple tissues. Interestingly, the chromosomal positions of allof the female-biased non-coding genes are in close proximity to protein-coding genes thatescape X-inactivation. This defines X-chromosome domains each of which contains a codingand a non-coding female-biased gene. Lack of repressive chromatin marks in non-codingtranscribed loci supports the possibility that they escape X-inactivation. Moreover, RNADNAcombined FISH experiments confirmed the biallelic expression of one such noveldomain. Conclusion: This study demonstrated that the amount of genes with sex-biased expression variesbetween individual brain regions in mouse. The sex-biased genes identified are localized onmany chromosomes. At the same time, sexually dimorphic gene expression that is common toseveral parts of the brain is mostly restricted to the sex chromosomes. Moreover, the studyuncovered multiple female-biased non-coding genes that are non-randomly co-localized onthe X-chromosome with protein-coding genes that escape X-inactivation. This raises thepossibility that expression of long non-coding RNAs may play a role in modulating geneexpression in domains that escape X-inactivation in mouse.
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