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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)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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| 4. |
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- 2019
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Journal article (peer-reviewed)
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| 5. |
- Altincekic, Nadide, et al.
(author)
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Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies
- 2024
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In: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 19:2, s. 563 - 574
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Journal article (peer-reviewed)abstract
- The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
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| 6. |
- Drake, Henrik, 1979, et al.
(author)
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Detecting the near-surface redox front in crystalline bedrock using fracture mineral distribution, geochemistry and U-series disequilibrium
- 2009
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In: Applied Geochemistry. ; 24:5, s. 1023-1039
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Journal article (peer-reviewed)abstract
- Oxidizing conditions normally prevail in surface waters and near-surface groundwaters, but there is usually a change to reducing conditions in groundwater at greater depth. Dissolved O2 originally present is consumed through biogenic and inorganic reactions along the flow paths. Fracture minerals participate in these reactions and the fracture mineralogy and geochemistry can be used to trace the redox front. An important task in the safety assessment of a potential repository for the disposal of nuclear waste in crystalline bedrock, at an approximate depth of 500 m in Sweden, is to demonstrate that reducing conditions can be maintained for a long period of time. Oxygen may damage the Cu canisters that host nuclear waste; additionally, in the event of a canister failure, oxidizing conditions may increase the mobility of some radionuclides. The present study of the near-surface redox front is based on mineralogical (redox-sensitive minerals), geochemical (redox-sensitive elements) and U-series disequilibrium investigations of mineral coatings along open fractures. The fractures have been sampled along drill cores from closely spaced, 100 m deep boreholes, which were drilled during the site investigation work in the Laxemar area, south-eastern Sweden, carried out by the Swedish Nuclear Fuel and Waste Management Co. (SKB). The distribution of the redox-sensitive minerals pyrite and goethite in open fractures shows that the redox front (switch from mainly goethite to mainly pyrite in the fractures) generally occurs at about 15-20 m depth. Calcite leaching by recharging water is indicated in the upper 20-30 m and positive Ce-anomalies suggest oxidation of Ce down to 20 m depth. The U-series radionuclides show disequilibrium in most of the samples, indicating mobility of U during the last 1 Ma. In the upper 20 m, U is mainly removed (due to oxidation) or has experienced complex removal and/or deposition. At depths of 35-55 m, both deposition and removal of U are indicated. Below 55 m, recent deposition of U is generally indicated which suggests removal of U near surface (oxidation) and deposition of U below the redox front. Scattered goethite occurrences below the general redox front (down to ca 80 m) and signs of U removal at 35-55 m mostly correlate with sections of high transmissivity (and/or high fracture frequencies). This shows that highly transmissive fractures are generally required to allow oxygenated groundwaters at depth greater than ca 30 m. Removal of U (oxidation) below 55 m within the last 300 ka is not observed. Although penetration of glacial waters to great depths has been confirmed in the study area, their potential O2 load seems to have been reduced near the surface.
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| 7. |
- Sandström, Björn, 1976, et al.
(author)
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Fracture mineralogy of the Forsmark site. SDM Forsmark : SKB R-08-102
- 2008
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Reports (other academic/artistic)abstract
- Detailed investigations of the fracture mineralogy and altered wall rock have been carried out as part of the site characterisation programme between 2003 and 2007 at Forsmark. The results have been published in a number of P-reports and in contributions to scientific journals. This report summarises and evaluates the data obtained during the detailed fracture mineralogical studies. The report includes descriptions of the identified fracture minerals and their chemical composition. A sequence of fracture mineralisations has been distinguished and provides information of the low to moderate temperature (brittle) geological and hydrogeological evolution at the site. Special focus has been paid to the chemical and stable isotopic composition of calcite to obtain palaeohydrogeological information. Chemical analyses of bulk fracture filling material have been carried out to identify possible sinks for certain elements and also to reveal the presence of minor phases rich in certain elements which have not been possible to detect by X-ray diffraction (XRD). Statistical analysis of the mineralogy in fractures outside deformation zones (i.e. within fracture domains FFM01, FFM02, FFM03 and FFM06) have been carried out concerning variation of fracture mineral distribution at depth and in different fracture domains. Uranium contents and uranium-series isotopes have been analysed on fracture coating material from hydraulically conductive fractures. Such analyses are also available from the groundwaters and the results are combined in order to reveal recent (< 1 Ma) removal/deposition of uranium in the fracture system. The redox conditions in the fracture system have been evaluated based on mineralogical and chemical indicators as well as Mössbauer analyses
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