| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Buell, Alexander K., et al.
(författare)
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Solution conditions determine the relative importance of nucleation and growth processes in alpha-synuclein aggregation
- 2014
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:21, s. 7671-7676
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Tidskriftsartikel (refereegranskat)abstract
- The formation of amyloid fibrils by the intrinsically disordered protein alpha-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that alpha-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of alpha-synuclein aggregation and aggregate spreading in the context of Parkinson disease.
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| 3. |
- Gaspar, Ricardo, et al.
(författare)
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Anomalous Salt Dependence Reveals an Interplay of Attractive and Repulsive Electrostatic Interactions in α-synuclein Fibril Formation
- 2020
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Ingår i: Quarterly Reviews in Biophysics Discovery. - : Cambridge University Press (CUP). - 2633-2892. ; 1
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Tidskriftsartikel (refereegranskat)abstract
- α-Synuclein (α-syn) is an intrinsically disordered protein with a highly asymmetric charge distribution, whose aggregation is linked to Parkinson’s disease. The effect of ionic strength was investigated at mildly acidic pH (5.5) in the presence of catalytic surfaces in the form of α-syn seeds or anionic lipid vesicles using thioflavin T fluorescence measurements. Similar trends were observed with both surfaces: increasing ionic strength reduced the rate of α-syn aggregation although the surfaces as well as α-syn have a net negative charge at pH 5.5. This anomalous salt dependence implies that short-range attractive electrostatic interactions are critical for secondary nucleation as well as heterogeneous primary nucleation. Such interactions were confirmed in Monte Carlo simulations of α-syn monomers interacting with surface-grafted C-terminal tails, and found to be weakened in the presence of salt. Thus, nucleation of α-syn aggregation depends critically on an attractive electrostatic component that is screened by salt to the extent that it outweighs the screening of the long-range repulsion between negatively charged monomers and negative surfaces. Interactions between the positively charged N-termini of α-syn monomers on the one hand, and the negatively C-termini of α-syn on fibrils or vesicles surfaces on the other hand, are thus critical for nucleation.
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| 4. |
- Gaspar, Ricardo, et al.
(författare)
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Eye lens crystallin proteins inhibit the autocatalytic amyloid amplification nature of mature α-synuclein fibrils
- 2020
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 15:6
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson´s disease is characterized by the accumulation of proteinaceous aggregates in Lewy bodies and Lewy Neurites. The main component found in such aggregates is α-synuclein. Here, we investigate how bovine eye lens crystallin proteins influence the aggregation kinetics of α-synuclein at mildly acidic pH (5.5) where the underlying aggregation mechanism of this protein is dominated by secondary nucleation of monomers on fibril surface providing an autocatalytic amyloid amplification process. Bovine α-, βH- and γB-crystallins were found to display chaperone-like activity inhibiting α-synuclein aggregation. This effect was shown to be time-dependent, with early additions of α-crystallin capable of retarding and even inhibiting aggregation during the time frame of the experiment. The inhibitory nature of crystallins was further investigated using trap and seed kinetic experiments. We propose crystallins interact with mature α-synuclein fibrils, possibly binding along the surfaces and at fibril free ends, inhibiting both elongation and monomer-dependent secondary nucleation processes in a mechanism that may be generic to some chaperones that prevent the onset of protein misfolding related pathologies.
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| 5. |
- Gaspar, Ricardo, et al.
(författare)
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Ganglioside lipids accelerate α-synuclein amyloid formation
- 2018
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1866:10, s. 1062-1072
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Tidskriftsartikel (refereegranskat)abstract
- The deposition of α-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of α-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of α-synuclein monomers and catalyse their aggregation at mildly acidic conditions. Although the extent of this catalytic effect was slightly higher for gangliosides, the results imply that charge interactions are more important than headgroup chemistry in triggering aggregation. In support of this idea, uncharged lipids with large headgroups were not found to induce any conformational change and only weakly catalyse aggregation. Intriguingly, aggregation was also triggered by free ganglioside headgroups, while these caused no conformational change of α-synuclein monomers. Our data reveal that partially folded α-synuclein helical intermediates are not required species in triggering of α-synuclein aggregation.
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| 6. |
- Gaspar, Ricardo, et al.
(författare)
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Secondary nucleation of monomers on fibril surface dominates α-synuclein aggregation and provides autocatalytic amyloid amplification
- 2017
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Ingår i: Quarterly Reviews of Biophysics. - 0033-5835. ; 50
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson's disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containing α-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration. The molecular mechanism underlying this process could be nucleation of monomers on fibril surfaces or fibril fragmentation. Here, we aim to distinguish between these mechanisms. The nature of the secondary processes was investigated using differential sedimentation analysis, trap and seed experiments, quartz crystal microbalance experiments and super-resolution microscopy. The results identify secondary nucleation of monomers on the fibril surface as the dominant secondary process leading to rapid generation of new aggregates, while no significant contribution from fragmentation was found. The newly generated oligomeric species quickly elongate to further serve as templates for secondary nucleation and this may have important implications in the spreading of PD.
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| 7. |
- Gaspar, Ricardo, et al.
(författare)
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Transient Lipid-Protein Structures and Selective Ganglioside Uptake During α-Synuclein-Lipid Co-aggregation
- 2021
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Ingår i: Frontiers in Cell and Developmental Biology. - : Frontiers Media SA. - 2296-634X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- α-Synuclein is a membrane-interacting protein involved in Parkinson’s disease. Here we have investigated the co-association of α-synuclein and lipids from ganglioside-containing model membranes. Our study relies on the reported importance of ganglioside lipids, which are found in high amounts in neurons and exosomes, on cell-to-cell prion-like transmission of misfolded α-synuclein. Samples taken along various stages of the aggregation process were imaged using cryogenic transmission electron microscopy, and the composition of samples corresponding to the final state analyzed using NMR spectroscopy. The combined data shows that α-synuclein co-assembles with lipids from the ganglioside (GM1)-containing model membranes. The lipid-protein samples observed during the aggregation process contain non-vesicular objects not present at the final stage, thus capturing the co-existence of species under non-equilibrium conditions. A range of different lipid-protein co-assemblies are observed during the time course of the reaction and some of these appear to be transient assemblies that evolve into other co-aggregates over time. At the end of the aggregation reaction, the samples become more homogeneous, showing thin fibrillar structures heavily decorated with small vesicles. From the NMR analysis, we conclude that the ratio of GM1 to phosphatidyl choline (PC) in the supernatant of the co-aggregated samples is significantly reduced compared to the GM1/PC ratio of the lipid dispersion from which these samples were derived. Taken together, this indicates a selective uptake of GM1 into the fibrillar aggregates and removal of GM1-rich objects from the solution.
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| 8. |
- Gaspar, Ricardo
(författare)
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α-Synuclein Fibril Formation and the Effects of Lipid Membranes
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyloid protein aggregation results in major disturbances of cellular processes in humans. The most common amyloid-related disorders are Alzheimer´s disease and Parkinson´s disease. Parkinson´s disease is charaterized by the formation of protein-rich aggregates that are deposited in neurons, termed Lewy bodies and Lewy neurites. The main component identified in these protein-rich deposits is the peptide α-synuclein. The work described here is mainly focused on in vitro aggregation studies of α-synuclein alone and in the presence of lipid membranes of different compositions. This is possible as we have identified conditions leading to reproducible thioflavin-T aggregation kinetics. The aggregation mechanism of α-synuclein was shown to be pH dependent. At neutral pH, both homogeneous primary nucleation and secondary processes are undetectable. At mildly acidic pH, present in the lumens of some intracellular compartments, secondary processes are enhanced. Experiments were performed to identify the nature of these secondary processes, distinguishing between fragmentation of fibrils and nucleation of monomers on the surface of existing aggregates. It was found that monomer-dependent secondary nucleation is the underlying dominant microscopic event of the aggregation process at mildly acidic pH, providing an autocatalytic amyloid amplification event. Crystallin chaperones purified from bovine eye lens were shown capable of inhibiting fibril formation of α-synuclein by affecting both secondary nucleation and elongation processes.In amyloid plaques associated with several amyloidogenic diseases, tightly associated lipids have been identified. We explore how lipid membranes interfere with the aggregation mechanism of α-synuclein. Our results show a dependence of the aggregation rate on lipid composition and lipid charge. Biological exosomes, isolated from neuroblastoma cells, were shown to accelerate α-synuclein aggregation. This effect was found to be due to neuro-specific ganglioside lipids. Investigating the catalytic effect of anionic lipid membranes on α-synuclein aggregation, it was shown that charge-based interactions, induce conformational change of α-synuclein from random coil to α-helix, and is also associated with rapid aggregation kinetics. However, charge-independent interactions are also important, as lipid membranes containing uncharged lipid species with large hydrophilic headgroups were shown capable of triggering α-synuclein aggregation, although far less efficiently and without inducing α-synuclein conformational change to α-helix. Investigating α-synuclein-ganglioside co-aggregation by imaging with cryo-EM at different time points along the aggregation reaction revealed aggregates with different structural morphology compared to those formed from pure α-synuclein. The images show a clear evolution of the vesicle size distribution, with a dramatic decrease in vesicle size during the lagphase of the aggregation reaction. Co-aggregates are thin and curly, decorated with small and monodisperse lipid vesicles contrary to compact bundles observed for fibrils formed from protein alone.
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| 9. |
- Grey, Marie, et al.
(författare)
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Acceleration of α-synuclein aggregation by exosomes
- 2015
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 290:5, s. 2969-2982
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Tidskriftsartikel (refereegranskat)abstract
- Exosomes are small vesicles released from cells into extra-cellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins flotillin-1 and alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as low concentration of preformed α-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effect of exosomes derived from naive cells and cells that over-express α-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry we found several phospholipid classes in the exosomes, including phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how α-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets.
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| 10. |
- Ribeiro, Maria Margarida, et al.
(författare)
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Genetic diversity and divergence at the Arbutus unedo L. (Ericaceae) westernmost distribution limit
- 2017
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:4
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Tidskriftsartikel (refereegranskat)abstract
- Mediterranean forests are fragile ecosystems vulnerable to recent global warming and reduction of precipitation, and a long-term negative effect is expected on vegetation with increasing drought and in areas burnt by fires. We investigated the spatial distribution of genetic variation of Arbutus unedo in the western Iberia Peninsula, using plastid markers with conservation and provenance regions design purposes. This species is currently undergoing an intense domestication process in the region, and, like other species, is increasingly under the threat from climate change, habitat fragmentation and wildfires. We sampled 451 trees from 15 natural populations from different ecological conditions spanning the whole species' distribution range in the region. We applied Bayesian analysis and identified four clusters ( north, centre, south, and a single-population cluster). Hierarchical AMOVA showed higher differentiation among clusters than among populations within clusters. The relatively low within-clusters differentiation can be explained by a common postglacial history of nearby populations. The genetic structure found, supported by the few available palaeobotanical records, cannot exclude the hypothesis of two independent A. unedo refugia in western Iberia Peninsula during the Last Glacial Maximum. Based on the results we recommend a conservation strategy by selecting populations for conservation based on their allelic richness and diversity and careful seed transfer consistent with current species' genetic structure.
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