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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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- Church, Deanna M, et al.
(författare)
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Lineage-specific biology revealed by a finished genome assembly of the mouse
- 2009
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 7:5, s. e1000112-
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Tidskriftsartikel (refereegranskat)abstract
- The mouse (Mus musculus) is the premier animal model for understanding human disease and development. Here we show that a comprehensive understanding of mouse biology is only possible with the availability of a finished, high-quality genome assembly. The finished clone-based assembly of the mouse strain C57BL/6J reported here has over 175,000 fewer gaps and over 139 Mb more of novel sequence, compared with the earlier MGSCv3 draft genome assembly. In a comprehensive analysis of this revised genome sequence, we are now able to define 20,210 protein-coding genes, over a thousand more than predicted in the human genome (19,042 genes). In addition, we identified 439 long, non-protein-coding RNAs with evidence for transcribed orthologs in human. We analyzed the complex and repetitive landscape of 267 Mb of sequence that was missing or misassembled in the previously published assembly, and we provide insights into the reasons for its resistance to sequencing and assembly by whole-genome shotgun approaches. Duplicated regions within newly assembled sequence tend to be of more recent ancestry than duplicates in the published draft, correcting our initial understanding of recent evolution on the mouse lineage. These duplicates appear to be largely composed of sequence regions containing transposable elements and duplicated protein-coding genes; of these, some may be fixed in the mouse population, but at least 40% of segmentally duplicated sequences are copy number variable even among laboratory mouse strains. Mouse lineage-specific regions contain 3,767 genes drawn mainly from rapidly-changing gene families associated with reproductive functions. The finished mouse genome assembly, therefore, greatly improves our understanding of rodent-specific biology and allows the delineation of ancestral biological functions that are shared with human from derived functions that are not.
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- Benner, J. S., et al.
(författare)
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A novel programme to evaluate and communicate 10-year risk of CHD reduces predicted risk and improves patients' modifiable risk factor profile
- 2008
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Ingår i: International Journal of Clinical Practice. - : Hindawi Limited. - 1742-1241 .- 1368-5031. ; 62:10, s. 1484-1498
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Tidskriftsartikel (refereegranskat)abstract
- Aims: We assessed whether a novel programme to evaluate/communicate predicted coronary heart disease (CHD) risk could lower patients' predicted Framingham CHD risk vs. usual care. Methods: The Risk Evaluation and Communication Health Outcomes and Utilization Trial was a prospective, controlled, cluster-randomised trial in nine European countries, among patients at moderate cardiovascular risk. Following baseline assessments, physicians in the intervention group calculated patients' predicted CHD risk and were instructed to advise patients according to a risk evaluation/communication programme. Usual care physicians did not calculate patients' risk and provided usual care only. The primary end-point was Framingham 10-year CHD risk at 6 months with intervention vs. usual care. Results: Of 1103 patients across 100 sites, 524 patients receiving intervention, and 461 receiving usual care, were analysed for efficacy. After 6 months, mean predicted risks were 12.5% with intervention, and 13.7% with usual care [odds ratio = 0.896; p = 0.001, adjusted for risk at baseline (17.2% intervention; 16.9% usual care) and other covariates]. The proportion of patients achieving both blood pressure and low-density lipoprotein cholesterol targets was significantly higher with intervention (25.4%) than usual care (14.1%; p < 0.001), and 29.3% of smokers in the intervention group quit smoking vs. 21.4% of those receiving usual care (p = 0.04). Conclusions: A physician-implemented CHD risk evaluation/communication programme improved patients' modifiable risk factor profile, and lowered predicted CHD risk compared with usual care. By combining this strategy with more intensive treatment to reduce residual modifiable risk, we believe that substantial improvements in cardiovascular disease prevention could be achieved in clinical practice.
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