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| 2. |
- 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. |
- Aguilar, Edgar A., et al.
(författare)
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Certifying an Irreducible 1024-Dimensional Photonic State Using Refined Dimension Witnesses
- 2018
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Ingår i: Physical Review Letters. - : AMER PHYSICAL SOC. - 0031-9007 .- 1079-7114. ; 120:23
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Tidskriftsartikel (refereegranskat)abstract
- We report on a new class of dimension witnesses, based on quantum random access codes, which are a function of the recorded statistics and that have different bounds for all possible decompositions of a high-dimensional physical system. Thus, it certifies the dimension of the system and has the new distinct feature of identifying whether the high-dimensional system is decomposable in terms of lower dimensional subsystems. To demonstrate the practicability of this technique, we used it to experimentally certify the generation of an irreducible 1024-dimensional photonic quantum state. Therefore, certifying that the state is not multipartite or encoded using noncoupled different degrees of freedom of a single photon. Our protocol should find applications in a broad class of modern quantum information experiments addressing the generation of high-dimensional quantum systems, where quantum tomography may become intractable.
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| 4. |
- Baloch, Ramen Munir, et al.
(författare)
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Indoor air pollution, physical and comfort parameters related to schoolchildren's health : Data from the European SINPHONIE study
- 2020
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Ingår i: Science of the Total Environment. - : ELSEVIER. - 0048-9697 .- 1879-1026. ; 739
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Tidskriftsartikel (refereegranskat)abstract
- Substantial knowledge is available on the association of the indoor school environment and its effect among schoolchildren. In the same context, the SINPHONIE (School indoor pollution and health: Observatory network in Europe) conducted a study to collect data and determine the distribution of several indoor air pollutants (IAPs), physical and thermal parameters and their association with eye, skin, upper-, lower respiratory and systemic disorder symptoms during the previous three months. Finally, data from 115 schools in 54 European cities from 23 countries were collected and included 5175 schoolchildren using a harmonized and standardized protocol. The association between exposures and the health outcomes were examined using logistic regression models on the environmental stressors assessed in classroom while adjusting for several confounding factors; a VOC (volatile organic compound) score defined as the sum of the number of pollutants to which the children were highly exposed (concentration > median of the distribution) in classroom was also introduced to evaluate the mul tiexposu re - outcome association. Schoolchildren while adjusting for several confounding factors. Schoolchildren exposed to above or equal median concentration of PM2.5, benzene, limonene, ozone and radon were at significantly higher odds of suffering from upper, lower airways, eye and systemic disorders. Increased odds were also observed for any symptom (sick school syndrome) among schoolchildren exposed to concentrations of limonene and ozone above median values. Furthermore, the risks for upper and lower airways and systemic disorders significantly increased with the VOCs score. Results also showed that increased ventilation rate was significantly associated with decreased odds of suffering from eye and skin disorders whereas similar association was observed between temperature and upper airways symptoms. The present study provides evidence that exposure to IAPs in schools is associated with various health problems in children. Further investigations are needed to confirm our findings. (C) 2020 Elsevier B.V. All rights reserved.
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| 5. |
- Biurrun, Idoia, et al.
(författare)
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Benchmarking plant diversity of Palaearctic grasslands and other open habitats
- 2021
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Ingår i: Journal of Vegetation Science. - Oxford : John Wiley & Sons. - 1100-9233 .- 1654-1103. ; 32:4
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Tidskriftsartikel (refereegranskat)abstract
- Journal of Vegetation Science published by John Wiley & Sons Ltd on behalf of International Association for Vegetation Science.Aims: Understanding fine-grain diversity patterns across large spatial extents is fundamental for macroecological research and biodiversity conservation. Using the GrassPlot database, we provide benchmarks of fine-grain richness values of Palaearctic open habitats for vascular plants, bryophytes, lichens and complete vegetation (i.e., the sum of the former three groups). Location: Palaearctic biogeographic realm. Methods: We used 126,524 plots of eight standard grain sizes from the GrassPlot database: 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 and 1,000 m2 and calculated the mean richness and standard deviations, as well as maximum, minimum, median, and first and third quartiles for each combination of grain size, taxonomic group, biome, region, vegetation type and phytosociological class. Results: Patterns of plant diversity in vegetation types and biomes differ across grain sizes and taxonomic groups. Overall, secondary (mostly semi-natural) grasslands and natural grasslands are the richest vegetation type. The open-access file ”GrassPlot Diversity Benchmarks” and the web tool “GrassPlot Diversity Explorer” are now available online (https://edgg.org/databases/GrasslandDiversityExplorer) and provide more insights into species richness patterns in the Palaearctic open habitats. Conclusions: The GrassPlot Diversity Benchmarks provide high-quality data on species richness in open habitat types across the Palaearctic. These benchmark data can be used in vegetation ecology, macroecology, biodiversity conservation and data quality checking. While the amount of data in the underlying GrassPlot database and their spatial coverage are smaller than in other extensive vegetation-plot databases, species recordings in GrassPlot are on average more complete, making it a valuable complementary data source in macroecology. © 2021 The Authors.
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| 6. |
- Díaz-Ramos, L. Aranzazú, et al.
(författare)
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Difference in the action spectra for UVR8 monomerisation and HY5 transcript accumulation in Arabidopsis
- 2018
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Ingår i: Photochemical and Photobiological Sciences. - : Royal Society of Chemistry. - 1474-905X .- 1474-9092. ; 17:8, s. 1108-1117
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Tidskriftsartikel (refereegranskat)abstract
- The photoreceptor UV RESISTANCE LOCUS 8 (UVR8) activates photomorphogenic responses when plants are exposed to ultraviolet-B (UVB) light. However, whereas the absorption spectrum of UVR8 peaks at 280 nm, action spectra for several photomorphogenic UV-B responses show maximal photon effectiveness at 290-300 nm. To investigate this apparent discrepancy we measured the effectiveness of UV wavelengths in initiating two responses in Arabidopsis: photoconversion of homodimeric UVR8 into the monomeric form, which is active in signaling, and accumulation of transcripts of the ELONGATED HYPOCOTYL 5 (HY5) transcription factor, which has a key role in UVR8-mediated responses. When purified UVR8 or Arabidopsis leaf extracts were exposed to UV light monomerisation was maximal at approximately 280 nm, which correlates with the UVR8 absorption spectrum. When intact plants were exposed to UV, monomerisation was most strongly initiated at approximately 290 nm, and this shift in maximal effectiveness could be explained by strong absorption or reflectance at 280 nm by leaf tissue. Notably, the action spectrum for accumulation of HY5 transcripts in the same leaf tissue samples used to assay UVR8 dimer/monomer status peaked at approximately 300 nm. Possible reasons for the difference in maximal photon effectiveness of UVR8 monomerisation and HY5 transcript accumulation in leaf tissue are discussed.
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| 7. |
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| 8. |
- Farkas, Daniel, et al.
(författare)
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Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding
- 2011
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Ingår i: Protein Expression and Purification. - San Diego : Academic Press. - 1046-5928 .- 1096-0279. ; 78:2, s. 156-166
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Tidskriftsartikel (refereegranskat)abstract
- The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I Ref. [5] and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and 15N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.
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| 9. |
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| 10. |
- Farkas, Daniel, 1980
(författare)
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Regulation of electron donation to photosystem 1
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Photosynthesis is the process where energy from light is used to catalyze the formation of energy-rich molecules such as NADPH and ATP. These reactions are in fact the result of a long series of electron-transfer reactions where water molecules are the source of electrons and NADP+ is the terminal electron acceptor. A consequence of the electron-transfer reactions is the translocation of protons across a phospholipid membrane. This proton gradient in turn drives ATP synthase to catalyze the formation of ATP from ADP. From this chain of events, molecular oxygen is a byproduct, leading to the oxygen-rich atmosphere we live in today. In this thesis, the objective has been to study the specific protein-protein interaction between the electron-donor protein plastocyanin (Pc) and the photosystem 1 (PSI) subunit PsaF. The physiological regulation of this interaction has been a major subject and hence should be kept in mind to understand the purpose of the different papers and studies reported here. In paper I we report on the cloning, expression and characterization of the luminal domain of spinach PsaF. Characterization by several different biophysical techniques revealed a protein domain which is very dynamic and consistent with molten-globule like structural features. A disulphide bridge formed between cysteine residues 8 and 63 appeared to have a major role in stabilizing the tertiary fold of this domain. Site-directed mutagenesis and zero-length cross-linking revealed a native-like interaction with Pc, strongly dependent on the electrostatic character of the two proteins. The findings in paper I led us to investigate whether light-induced changes in the Mg(II) content in the chloroplast lumen can modulate the electron donation to PSI, in particular the electrostatic interaction between Pc and PsaF. Using NMR and EPR spectroscopy to characterize the Pc-PsaF complex and the one formed between Pc and Mg(II) we could observe similar binding constants. A competitive effect could be observed for the binding of Mg(II) to the Pc-PsaF complex, hence suggesting that the two interact with the same region of Pc. By studying the paramagnetic relaxation enhancement of the Mg(II) analogue, Mn(II), and its effect on Pc’s 15N-HSQC spectra, we could calculate the structure of the transient Pc-Mn(II) complex. The results presented suggest a specific binding site for Mg(II) that may regulate the binding of Pc to PSI in vivo. In paper III, we show that the luminal domain of PsaF is a target for thioredoxin-mediated reduction of the disulphide bridge formed between cysteines 8 and 63. Furthermore, we show that the thiolated form of PsaF has a lower affinity towards reduced Pc than when the disulfide bridge is intact. Time-resolved absorbance measurements and fluorescence electrophoresis show that oxidized Pc can re-oxidize PsaF and thus restore the active form of this domain. The PSI subunit PsaN is a weak modulator of the electron donation from Pc to PSI. In paper IV we present a methodology for the recombinant production of this protein subunit. Problems with unspecific proteolysis and degradation by Escherichia coli proteases are tackled.
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