| 1. |
- 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. |
- Guo, J. -H, et al.
(författare)
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Electronic structure study of the bases in DNA duplexes by in situ photon-in/photon-out soft X-ray spectroscopy
- 2010
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Ingår i: Journal of Electron Spectroscopy and Related Phenomena. - : Elsevier BV. - 0368-2048 .- 1873-2526. ; 181:2-3, s. 197-201
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Tidskriftsartikel (refereegranskat)abstract
- Understanding protein functionality is of fundamental importance in biochemistry. Soft X-ray transitions, where the core-level vacancies are filled by the valence-orbital electrons, give direct information about the chemical bonding. Soft X-ray absorption and emission study of poly(dG) -poly(dC) in aqueous solutions can elucidate the relation between the structure and functionality of proteins. We report the N K-edge soft X-ray absorption (XAS) and resonant soft X-ray emission spectroscopy (XES) to characterize the electronic structure near the Fermi level of DNA duplexes to specify the charge migration mechanism. Since N atoms are included in only bases in DNA duplexes, the XES spectra excited from N Is to unoccupied states purely extract the electronic orbital features of the bases in DNA. The fact that N atoms in different bonding environments form well-defined structure has been determined. The experimental findings provide the evidence for the charge-hopping and/or charge-transfer effects in understanding of electric conduction in DNA duplexes when electrons pass through the pi* states of DNA bases.
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| 3. |
- Balasubramanian, Padmanabhan, et al.
(författare)
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Valence band electronic structure of Nd1-xYxMnO3 using X-ray absorption, photoemission and GGA plus U calculations
- 2013
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Ingår i: Journal of Electron Spectroscopy and Related Phenomena. - : Elsevier BV. - 0368-2048 .- 1873-2526. ; 189, s. 51-55
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Tidskriftsartikel (refereegranskat)abstract
- The electronic structures of Nd1-xYxMnO3 (x=0-0.5) were studied using X-ray absorption near-edge structure (XANES) at the Mn L-3,L-2- and O K-edge along with valence-band photoemission spectroscopy (VB-PES). The systematic increase in white-line intensity of the Mn L-3,L-2-edge with doping, suggests a decrease in the occupancy of Mn 3d orbitals. The O K-edge XANES shows a depletion of unoccupied states above the Fermi energy. The changes in the O K-edge spectra due to doping reflects an increase in the Jahn-Teller distortion. The VB-PES shows broadening of the features associated with Mn 3d and O 2p hybridized states and the shift of these features to a slightly higher binding energy in agreement with our GGA + U calculations. The system shows a net shift of the occupied and unoccupied states away from the Fermi energy with doping. The shift in theoretical site-projected density of states of x=0.5 composition with respect to x=0 suggest a subtle change from a charge transfer to Mott-Hubbard type insulator. (C) 2013 Elsevier B.V. All rights reserved.
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| 4. |
- Lee, Po-Shun, et al.
(författare)
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mTORC1-S6K activation by endotoxin contributes to cytokine up-regulation and early lethality in animals.
- 2010
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 5:12
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Tidskriftsartikel (refereegranskat)abstract
- mTORC1 (mammalian target of rapamycin complex 1) activation has been demonstrated in response to endotoxin challenge, but the mechanism and significance are unclear. We investigated the effect of mTORC1 suppression in an animal model of endotoxemia and in a cellular model of endotoxin signaling.Mice were treated with the mTORC1 inhibitor rapamycin or vehicle prior to lethal endotoxin challenge. Mortality and cytokine levels were assessed. Cultured macrophage-like cells were challenged with endotoxin with or without inhibitors of various pathways known to be upstream of mTORC1. Activated pathways, including downstream S6K pathway, were assessed by immunoblots. We found that mTORC1-S6K suppression by rapamycin delayed mortality of mice challenged with lethal endotoxin, and was associated with dampened circulating levels of VEGF, IL-1β, IFN-γ and IL-5. Furthermore, in vitro cellular studies demonstrated that LPS (lipopolysaccharide) activation of mTORC1-S6K still occurs in the presence of PI3K-Akt inhibition alone, but can be suppressed by concurrent inhibition of PI3K-Akt and MEK-ERK pathways.We conclude that cellular activation of mTORC1-S6K contributes to cytokine up-regulation and mortality in response to endotoxin, and may occur via multiple pathways.
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| 5. |
- Rothfels, Carl J., et al.
(författare)
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Overcoming Deep Roots, Fast Rates, and Short Internodes to Resolve the Ancient Rapid Radiation of Eupolypod II Ferns
- 2012
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Ingår i: Systematic Biology. - : Oxford University Press (OUP). - 1063-5157 .- 1076-836X. ; 61:3, s. 490-509
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Tidskriftsartikel (refereegranskat)abstract
- Backbone relationships within the large eupolypod II clade, which includes nearly a third of extant fern species, have resisted elucidation by both molecular and morphological data. Earlier studies suggest that much of the phylogenetic intractability of this group is due to three factors: (i) a long root that reduces apparent levels of support in the ingroup; (ii) long ingroup branches subtended by a series of very short backbone internodes (the "ancient rapid radiation" model); and (iii) significantly heterogeneous lineage-specific rates of substitution. To resolve the eupolypod II phylogeny, with a particular emphasis on the backbone internodes, we assembled a data set of five plastid loci (atpA, atpB, matK, rbcL, and trnG-R) from a sample of 81 accessions selected to capture the deepest divergences in the clade. We then evaluated our phylogenetic hypothesis against potential confounding factors, including those induced by rooting, ancient rapid radiation, rate heterogeneity, and the Bayesian star-tree paradox artifact. While the strong support we inferred for the backbone relationships proved robust to these potential problems, their investigation revealed unexpected model-mediated impacts of outgroup composition, divergent effects of methods for countering the star-tree paradox artifact, and gave no support to concerns about the applicability of the unrooted model to data sets with heterogeneous lineage-specific rates of substitution. This study is among few to investigate these factors with empirical data, and the first to compare the performance of the two primary methods for overcoming the Bayesian star-tree paradox artifact. Among the significant phylogenetic results is the near-complete support along the eupolypod II backbone, the demonstrated paraphyly of Woodsiaceae as currently circumscribed, and the well-supported placement of the enigmatic genera Homalosorus, Diplaziopsis, and Woodsia.
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