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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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- Kennicutt, M. C., et al.
(author)
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Delivering 21st century Antarctic and Southern Ocean science
- 2016
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In: Antarctic Science. - 0954-1020 .- 1365-2079. ; 28, s. 407-423
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Journal article (peer-reviewed)abstract
- © Antarctic Science Ltd 2016.The Antarctic Roadmap Challenges (ARC) project identified critical requirements to deliver high priority Antarctic research in the 21st century. The ARC project addressed the challenges of enabling technologies, facilitating access, providing logistics and infrastructure, and capitalizing on international co-operation. Technological requirements include: i) innovative automated in situ observing systems, sensors and interoperable platforms (including power demands), ii) realistic and holistic numerical models, iii) enhanced remote sensing and sensors, iv) expanded sample collection and retrieval technologies, and v) greater cyber-infrastructure to process 'big data' collection, transmission and analyses while promoting data accessibility. These technologies must be widely available, performance and reliability must be improved and technologies used elsewhere must be applied to the Antarctic. Considerable Antarctic research is field-based, making access to vital geographical targets essential. Future research will require continent- and ocean-wide environmentally responsible access to coastal and interior Antarctica and the Southern Ocean. Year-round access is indispensable. The cost of future Antarctic science is great but there are opportunities for all to participate commensurate with national resources, expertise and interests. The scope of future Antarctic research will necessitate enhanced and inventive interdisciplinary and international collaborations. The full promise of Antarctic science will only be realized if nations act together.
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- Ausili, A., et al.
(author)
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Membrane docking mode of the C2 domain of PKCε: An infrared spectroscopy and FRET study
- 2013
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In: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736. ; 1828:2, s. 552-560
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Journal article (peer-reviewed)abstract
- The C2 domain of PKCε binds to negatively charged phospholipids but little is known so far about the docking orientation of this domain when it is bound. By using a FRET assay we have studied the binding of this domain to model membranes. We have also used ATR-Fourier transform infrared spectroscopy with polarized light (ATR-FTIR) to determine the docking mode by calculating the β-sandwich orientation when the domain is bound to different types of model membranes. The vesicle lipid compositions were: POPC/POPE/POPA (22:36:42) imitating the inner leaflet of a plasma membrane, POPC/POPA (50:50) in which POPE has been eliminated with respect to the former composition and POPC/POPE/CL (43:36:21) imitating the inner mitochondrial membrane. Results show that the β-sandwich of the PKCα-C2 domain is inclined at an angle α close to 45 to the membrane normal. Some differences were found with respect to the extent of binding as a function of phospholipid composition and small changes on secondary structure were only evident when the domain was bound to model membranes of POPC/POPA: in this case, the percentage of β-sheet of the C2 domain increases if compared with the secondary structure of the domain in the absence of vesicles. With respect to the β-sandwich orientation, when the domain is bound to POPC/POPE/CL membranes it forms an angle with the normal to the surface of the lipid bilayer (39) smaller than that one observed when the domain interacts with vesicles of POPC/POPA (49). © 2012 Elsevier B.V. All rights reserved.
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- Ausili, A., et al.
(author)
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Quartz crystal microbalance with dissipation monitoring and the real-time study of biological systems and macromolecules at interfaces
- 2012
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In: Biomedical Spectroscopy and Imaging. - 2212-8794 .- 2212-8808. ; 1:4, s. 325-338
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Journal article (peer-reviewed)abstract
- QCM-D technique is based on the physical phenomenon that generates an acoustic shear wave with an oscillating resonance in quartz resulting in an evanescent wave that arises at the interface of the quartz and the solution. The amplitude of the acoustic wave is influenced by the deposition of material onto the quartz surface and from the subsequent decrease of the frequency the bound mass can be calculated. The dissipation shift which arises inform about viscoelasticity and flexibility of the adsorbed material. QCM-D can be applied for real-time studies of several biological systems since it is a simple, fast, low-cost and sensitive technique without having to label any sample. Common applications in biological field include measurements on adsorption of lipids, proteins, DNA and cells directly onto the surface of the sensor, which generally are chemically modified by self-assembled monolayer (SAM) technique or by spin-coated polymers. QCM-D can also be used to study molecular interactions between macromolecules and adsorbed materials. Three examples of the use of this technique are presented, namely the docking orientation of the C2 domain of PKCε on phospholipid membranes, the conformational changes of fibrinogen adsorbed to model acrylic polymers and the attachment of endothelial cells to carboxylated polymers of different configuration.
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