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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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| 5. |
- Menard, J.E., et al.
(författare)
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Progress in understanding error-field physics in NSTX spherical torus plasmas
- 2010
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Ingår i: Nuclear Fusion. - : IOP Publishing. - 1741-4326 .- 0029-5515. ; 50:4, s. 045008-
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Tidskriftsartikel (refereegranskat)abstract
- The low-aspect ratio, low magnetic field and wide range of plasma beta of NSTX plasmas provide new insight into the origins and effects of magnetic field errors. An extensive array of magnetic sensors has been used to analyse error fields, to measure error-field amplification and to detect resistive wall modes (RWMs) in real time. The measured normalized error-field threshold for the onset of locked modes shows a linear scaling with plasma density, a weak to inverse dependence on toroidal field and a positive scaling with magnetic shear. These results extrapolate to a favourable error-field threshold for ITER. For these low-beta locked-mode plasmas, perturbed equilibrium calculations find that the plasma response must be included to explain the empirically determined optimal correction of NSTX error fields. In high-beta NSTX plasmas exceeding the n = 1 no-wall stability limit where the RWM is stabilized by plasma rotation, active suppression of n = 1 amplified error fields and the correction of recently discovered intrinsic n = 3 error fields have led to sustained high rotation and record durations free of low-frequency core MHD activity. For sustained rotational stabilization of the n = 1 RWM, both the rotation threshold and the magnitude of the amplification are important. At fixed normalized dissipation, kinetic damping models predict rotation thresholds for RWM stabilization to scale nearly linearly with particle orbit frequency. Studies for NSTX find that orbit frequencies computed in general geometry can deviate significantly from those computed in the high-aspect ratio and circular plasma cross-section limit, and these differences can strongly influence the predicted RWM stability. The measured and predicted RWM stability is found to be very sensitive to the E × B rotation profile near the plasma edge, and the measured critical rotation for the RWM is approximately a factor of two higher than predicted by the MARS-F code using the semi-kinetic damping model.
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| 7. |
- Yusuf, D, et al.
(författare)
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The transcription factor encyclopedia
- 2012
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Ingår i: Genome biology. - : Springer Science and Business Media LLC. - 1474-760X .- 1465-6906. ; 13:3, s. R24-
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Tidskriftsartikel (refereegranskat)
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| 9. |
- Chapin, F. Stuart, III, et al.
(författare)
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Ecosystem stewardship : sustainability strategies for a rapidly changing planet
- 2010
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Ingår i: Trends in Ecology & Evolution. - : Elsevier BV. - 0169-5347 .- 1872-8383. ; 25:4, s. 241-249
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Forskningsöversikt (refereegranskat)abstract
- Ecosystem stewardship is an action-oriented framework intended to foster the social ecological sustainability of a rapidly changing planet. Recent developments identify three strategies that make optimal use of current understanding in an environment of inevitable uncertainty and abrupt change: reducing the magnitude of, and exposure and sensitivity to, known stresses; focusing on proactive policies that shape change; and avoiding or escaping unsustainable social ecological traps. As we discuss here, all social ecological systems are vulnerable to recent and projected changes but have sources of adaptive capacity and resilience that can sustain ecosystem services and human well-being through active ecosystem stewardship.
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| 10. |
- Lee, Hyun Ju, et al.
(författare)
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Intricate role of water in proton transport through cytochrome c oxidase
- 2010
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 132:45, s. 16225-16239
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O2 to H2O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from Rhodobacter sphaeroides. Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules halfway up the D pathway, were replaced by more bulky hydrophobic residues (Ser200Ile, Ser200Val/Ser201Val, and Ser200Val/Ser201Tyr) to query the effects of changing the local structure on enzyme activity as well as proton uptake, release, and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcO's were lowered, both the Ser200Ile and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the PR → F and F → O transitions (PR is the "peroxy" intermediate formed at the catalytic site upon reaction of the four-electron-reduced CytcO with O2, F is the oxoferryl intermediate, and O is the fully oxidized CytcO). Furthermore, the PR → F transition is shown to be essentially pH independent up to pH 12 (i.e., the apparent pKa of Glu286 is increased from 9.4 by at least 3 pKa units) in the Ser200Val/Ser201Val mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dynamics can cause intriguing energetic consequences and hence provide mechanistic insights that would never be detected in static structures or simulations of the system with fixed protonation states (i.e., lacking explicit proton transport). The results are discussed in terms of the proton-pumping mechanism of CytcO.
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