| 1. |
- 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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| 2. |
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| 3. |
- 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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| 4. |
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| 5. |
- 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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| 8. |
- Nyikó, Tünde, et al.
(författare)
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Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex
- 2013
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 41:13, s. 6715-28
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Tidskriftsartikel (refereegranskat)abstract
- Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3'untranslated region (UTR). In yeasts, unusually long 3'UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3'UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3'UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD.
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| 9. |
- Vasefi, Fartash, et al.
(författare)
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Polarization-Sensitive Hyperspectral Imaging in vivo : A Multimode Dermoscope for Skin Analysis
- 2014
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 4:1
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Tidskriftsartikel (refereegranskat)abstract
- Attempts to understand the changes in the structure and physiology of human skin abnormalities by non-invasive optical imaging are aided by spectroscopic methods that quantify, at the molecular level, variations in tissue oxygenation and melanin distribution. However, current commercial and research systems to map hemoglobin and melanin do not correlate well with pathology for pigmented lesions or darker skin. We developed a multimode dermoscope that combines polarization and hyperspectral imaging with an efficient analytical model to map the distribution of specific skin bio-molecules. This corrects for the melanin-hemoglobin misestimation common to other systems, without resorting to complex and computationally intensive tissue optical models. For this system's proof of concept, human skin measurements on melanocytic nevus, vitiligo, and venous occlusion conditions were performed in volunteers. The resulting molecular distribution maps matched physiological and anatomical expectations, confirming a technologic approach that can be applied to next generation dermoscopes and having biological plausibility that is likely to appeal to dermatologists.
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| 10. |
- Vasefi, Fartash, et al.
(författare)
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Quantifying the optical properties and chromophore concentrations of turbid media using polarization sensitive hyperspectral imaging : optical phantom studies
- 2013
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Ingår i: Proceedings Volume 8587, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI; 85870Z (2013) SPIE BiOS, 2013, San Francisco, California, United States. - : SPIE - International Society for Optical Engineering.
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Konferensbidrag (refereegranskat)abstract
- We present a polarization-sensitive hyperspectral imaging system (SkinSpect) that employs a spectrally-programmable light source in the visible and NIR domains. Multiple tissue-mimicking phantoms were fabricated to mimic the optical properties of normal skin as well as pigmented light and dark moles. The phantoms consist of titanium dioxide and a mixture of coffee, red food dye, and naphthol green as the scattering and the three absorptive agents in a polydimethylsiloxane (PDMS) base. Phantoms were produced with both smooth and rough textured surfaces and tested using Spatial Frequency Domain Imaging (SFDI) and Spatially Modulated Quantitative Spectroscopy (SMoQS) for homogeneity as well as determining absorption and scattering variance, respectively. The reflectance spectral images were also recorded using the SkinSpect research prototype; the spectral signatures of the phantoms were calculated using a two-flux single-layer Kubelka-Munk model and non-negative least square fitting routine was applied to extract the relative concentrations of the individual phantom components.
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