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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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| 3. |
- Agholme, Fredrik, et al.
(författare)
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Anti-sclerostin antibody and mechanical loading appear to influence metaphyseal bone independently in rats
- 2011
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Ingår i: Acta Orthopaedica. - : Taylor and Francis. - 1745-3674 .- 1745-3682. ; 82:5, s. 628-632
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Tidskriftsartikel (refereegranskat)abstract
- Background and purpose: Sclerostin is produced by osteocytes and is an inhibitor of bone formation. Thus, inhibition of sclerostin by a monoclonal antibody increases bone formation and improves fracture repair. Sclerostin expression is upregulated in unloaded bone and is downregulated by loading. We wanted to determine whether an anti-sclerostin antibody would stimulate metaphyseal healing in unloaded bone in a rat model.Methods: 10-week-old male rats (n = 48) were divided into 4 groups, with 12 in each. In 24 rats, the right hind limb was unloaded by paralyzing the calf and thigh muscles with an injection of botulinum toxin A (Botox). 3 days later, all the animals had a steel screw inserted into the right proximal tibia. Starting 3 days after screw insertion, either anti-sclerostin antibody (Scl-Ab) or saline was given twice weekly. The other 24 rats did not receive Botox injections and they were treated with Scl-Ab or saline to serve as normal-loaded controls. Screw pull-out force was measured 4 weeks after insertion, as an indicator of the regenerative response of bone to trauma.Results: Unloading reduced the pull-out force. Scl-Ab treatment increased the pull-out force, with or without unloading. The response to the antibody was similar in both groups, and no statistically significant relationship was found between unloading and antibody treatment. The cancellous bone at a distance from the screw showed changes in bone volume fraction that followed the same pattern as the pull-out force.Interpretation: Scl-Ab increases bone formation and screwfixation to a similar degree in loaded and unloaded bone.
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| 4. |
- Agholme, Fredrik, et al.
(författare)
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Sclerostin Antibody Treatment Enhances Metaphyseal Bone Healing in Rats
- 2010
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Ingår i: JOURNAL OF BONE AND MINERAL RESEARCH. - : American Society for Bone and Mineral Research. - 0884-0431. ; 25:11, s. 2412-2418
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Tidskriftsartikel (refereegranskat)abstract
- Sclerostin is the product of the SOST gene Loss of-function mutations in the SOST gene result in a high bone-mass phenotype demonstrating that sclerostin is a negative regulator of bone mass Primarily expressed by osteocytes in bone sclerostin is reported to bind the LRP5/6 receptor thereby antagonizing canonical Wnt signaling and negatively regulating bone formation We therefore investigated whether systemic administration of a sclerostin neutralizing antibody would increase the regeneration of traumatized metaphyseal bone in rats Young male rats had a screw inserted in the proximal tibia and were divided into six groups given 25 mg/kg of sclerostin antibody or control twice a week subcutaneously for 2 or 4 weeks In four groups, the screws were tested for pull out strength At the time of euthanasia a similar screw also was inserted in the contralateral tibia and pull-out tested immediately Sclerostin antibody significantly increased the pull out force by almost 50% compared with controls after 2 and 4 weeks Also the screws inserted at the time of euthanasia showed increased pull out force Micro-computed tomography (mu CT) of the remaining two groups showed that the antibody led to a 30% increase in bone volume fraction in a region surrounding the screw There also was a general increase in trabecular thickness in cancellous bone Thus as measured by the amount of bone and its mechanical resistance the sclerostin antibody increased bone formation during metaphyseal repair but also in untraumatized bone
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| 6. |
- Dida, Mulatu Geleta, et al.
(författare)
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Domestication of Lepidium campestre as part of Mistra Biotech, a research programme focused on agro biotechnology for sustainable food
- 2014
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Ingår i: Perennial Crops for Food Security Proceedings of the FAO Expert Workshop. - 9789251079980 - 9789251079997 ; , s. 141-147
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Bokkapitel (refereegranskat)abstract
- Several important challenges are facing agriculture . In the stride towards lowering the negative environmental impact of food production while maint aining and increasing the production, both basic farming practices and novel technologies are important tools. The use of biotechnology in breeding, however, is not uncontroversial. The crit icism has many angles and often relates to the applications brought forward by large, multinationa l companies, and farmers growing dependence on these companies when it comes to seeds. Question s are being raised about ethical acceptability, and about the health and environmental impacts. A g eneral aversion to what is often referred to as the “industrialization” of agriculture, and t o “unnaturalness”, also emerges in the debate. In the research programme referred to as Mistra Biotech , we include both philosophy and natural and social sciences. The overall goal is to facilitate production systems that are sustainable from ecological, social and economic perspectives. The objectives of the programme include developing: * new elite plant lines that have benefits for consumers, farmers, the food industry and the environment; * agribiotechnology tools that are important for achieving new product qualities, healthier crops and livestock, and for solving environmental problems in agriculture; * basis for sustainable production systems that contribute to increased competitiveness in Swedish agriculture and food production; * tools for ethical scrutiny of agricultural biotechnology that combine high demands on safety with encouragement of innovations; * basis for improved regulatory approaches and private-public relationships. The programme includes six component projects, in which domestication of a new biennial oilseed crop Lepidium campestre (field cress) is a major research focus. Questions we will try to answer within this programme include: Can biotechnology be used to improve crops which mitigate climate change or benefit the environment? What potential is there to commercialize such a crop? How would the consumers react to products made from it? Can breeding technology be improved further? Why does the market for genetically improved plant and animal materials look the way it does? What ethical concerns does the use of biotechnology raise? And how do all these issues feed into future agricultural systems? The results are integrated in the synthesis project called the Centre for Agriculture and Food Systems Analysis and Synthesis (AgriSA). Here we emphasise the research within Component Project1, with a focus on the domestication of field cress.
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| 8. |
- Guan, Rui, et al.
(författare)
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Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica
- 2014
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 12, s. 193-203
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Tidskriftsartikel (refereegranskat)abstract
- Erucic acid is a valuable industrial fatty acid with many applications. The main producers of this acid are today high erucic rapeseed (Brassica napus) and mustard (Brassica juncea), which have 45%-50% of erucic acid in their seed oils. Crambe abyssinica is an alternative promising producer of this acid as it has 55%-60% of erucic acid in its oil. Through genetic modification (GM) of three genes, we have previously increased the level of erucic acid to 71% (68mol%) in Crambe seed oil. In this study, we further investigated different aspects of oil biosynthesis in the developing GM Crambe seeds in comparison with wild-type (Wt) Crambe, rapeseed and safflower (Carthamus tinctorius). We show that Crambe seeds have very low phosphatidylcholine-diacylglycerol interconversion, suggesting it to be the main reason why erucic acid is limited in the membrane lipids during oil biosynthesis. We further show that GM Crambe seeds have slower seed development than Wt, accompanied by slower oil accumulation during the first 20days after flowering (DAF). Despite low accumulation of erucic acid during early stages of GM seed development, nearly 86mol% of all fatty acids accumulated between 27 and 50 DAF was erucic acid, when 40% of the total oil is laid down. Likely bottlenecks in the accumulation of erucic acid during early stages of GM Crambe seed development are discussed.
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| 9. |
- Hofvander, Per, et al.
(författare)
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Wax ester profiling of seed oil by nano-electrospray ionization tandem mass spectrometry
- 2013
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Ingår i: Plant Methods. - : Springer Science and Business Media LLC. - 1746-4811. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Conclusions: We developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.
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