| 1. |
- Lubart, Quentin, 1989, et al.
(author)
-
High throughput size-determination and multiplexed fluorescence analysis of single biological particles in a nanofluidic device
- 2019
-
In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. ; , s. 1420-1421
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Conference paper (peer-reviewed)abstract
- Biological nanoparticles, such as exosomes and viruses, are responsible for a multitude of important functions, but methods to characterize them on the single particle level are rare. We here present a nanofluidic platform for multi-parametric characterization of biological nanoparticles with high throughput. The device consists of feeding microchannels and an array of ~100 nanochannels where the nanoparticles can be characterized. We determine the size by analyzing the Brownian motion of the particles and quantify their content based on fluorescence imaging of up to three different colors. We successfully benchmark our method against existing techniques, such as Nanoparticle Tracking Analysis (NTA).
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| 2. |
- Lubart, Quentin, 1989, et al.
(author)
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Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO
- 2020
-
In: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 22:16, s. 8781-8790
-
Journal article (peer-reviewed)abstract
- Lipophilic carbocyanine dyes are widely used as fluorescent cell membrane probes in studies ranging from biophysics to cell biology. While they are extremely useful for qualitative observation of lipid structures, a major problem impairing quantitative studies is that the chemical environment of the lipid bilayer affects both the dye's insertion efficiency and photophysical properties. We present a systematic investigation of the sulphonated carbocyanine dye 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl) (SP-DiO) and demonstrate how its insertion efficiency into pre-formed lipid bilayers and its photophysical properties therein determine its apparent fluorescence intensity in different lipid environments. For this purpose, we use large unilamellar vesicles (LUVs) made of lipids with distinct chain unsaturation, acyl chain length, head group charge, and with variation in membrane cholesterol content as models. Using a combination of absorbance, fluorescence emission, and fluorescence lifetime measurements we reveal that SP-DiO incorporates more efficiently into liquid disordered phases compared to gel phases. Moreover, incorporation into the latter phase is most efficient when the mismatch between the length of the lipid and dye hydrocarbon chains is small. Furthermore, SP-DiO incorporation is less efficient in LUVs composed of negatively charged lipids. Lastly, when cholesterol was included in the LUV membranes, we observed significant spectral shifts, consistent with dye aggregation. Taken together, our study highlights the complex interplay between membrane composition and labeling efficiency with lipophilic dyes and advocates for careful assessment of fluorescence data when attempting a quantitative analysis of fluorescence data with such molecules.
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| 3. |
- Esbjörner, Elin K., et al.
(author)
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Membrane binding of pH-sensitive Influenza fusion peptides. Positioning, configuration and induced leakage in lipid vesicles models
- 2007
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:47, s. 13490-13504
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Journal article (peer-reviewed)abstract
- pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA 1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion (∼60-65° relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue
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| 4. |
- Friedrich, R., et al.
(author)
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A nano flow cytometer for single lipid vesicle analysis
- 2017
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In: Lab on a Chip - Miniaturisation for Chemistry and Biology. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 17:5, s. 830-841
-
Journal article (peer-reviewed)abstract
- We present a nanofluidic device for fluorescence-based detection and characterization of small lipid vesicles on a single particle basis. The device works like a nano flow cytometer where individual vesicles are visualized by fluorescence microscopy while passing through parallel nanochannels in a pressure-driven flow. An experiment requires less than 20 mu l sample volume to quantify both the vesicle content and the fluorescence signals emitted by individual vesicles. We show that the device can be used to accurately count the number of fluorescent synthetic lipid vesicles down to a vesicle concentration of 170 fM. We also show that the size-distribution of the vesicles can be resolved from their fluorescence intensity distribution after calibration. We demonstrate the applicability of the assay in two different examples. In the first, we use the nanofluidic device to determine the particle concentration in a sample containing cell-derived extracellular vesicles labelled with a lipophilic dye. In the second, we demonstrate that dual-color detection can be used to probe peptide binding to synthetic lipid vesicles; we identify a positive membrane-curvature sensing behavior of an arginine enriched version of the Antennapedia homeodomain peptide penetratin. Altogether, these results illustrate the potential of this nanofluidic-based methodology for characterization and quantification of small biological vesicles and their interactors without ensemble averaging. The device is therefore likely to find use as a quantitative analytical tool in a variety of fields ranging from diagnostics to fundamental biology research. Moreover, our results have potential to facilitate further development of automated lab-on-a-chip devices for vesicle analysis.
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| 5. |
- Halipi, Vesa, 1996, et al.
(author)
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Extracellular Vesicles Slow Down Aβ(1−42) Aggregation by Interfering with the Amyloid Fibril Elongation Step
- 2024
-
In: ACS Chemical Neuroscience. - 1948-7193. ; 15:5, s. 944-954
-
Journal article (peer-reviewed)abstract
- Formation of amyloid-β (Aβ) fibrils is a central pathogenic feature of Alzheimer's disease. Cell-secreted extracellular vesicles (EVs) have been suggested as disease modulators, although their exact roles and relations to Aβ pathology remain unclear. We combined kinetics assays and biophysical analyses to explore how small (<220 nm) EVs from neuronal and non-neuronal human cell lines affected the aggregation of the disease-associated Aβ variant Aβ(1−42) into amyloid fibrils. Using thioflavin-T monitored kinetics and seeding assays, we found that EVs reduced Aβ(1−42) aggregation by inhibiting fibril elongation. Morphological analyses revealed this to result in the formation of short fibril fragments with increased thicknesses and less apparent twists. We suggest that EVs may have protective roles by reducing Aβ(1−42) amyloid loads, but also note that the formation of small amyloid fragments could be problematic from a neurotoxicity perspective. EVs may therefore have double-edged roles in the regulation of Aβ pathology in Alzheimer's disease.
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| 6. |
- Jõemetsa, Silver, 1990, et al.
(author)
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Independent Size and Fluorescence Emission Determination of Individual Biological Nanoparticles Reveals that Lipophilic Dye Incorporation Does Not Scale with Particle Size
- 2020
-
In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 36:33, s. 9693-9700
-
Journal article (peer-reviewed)abstract
- Advancements in nanoparticle characterization techniques are critical for improving the understanding of how biological nanoparticles (BNPs) contribute to different cellular processes, such as cellular communication, viral infection, as well as various drug-delivery applications. Since BNPs are intrinsically heterogeneous, there is a need for characterization methods that are capable of providing information about multiple parameters simultaneously, preferably at the single-nanoparticle level. In this work, fluorescence microscopy was combined with surface-based two-dimensional flow nanometry, allowing for simultaneous and independent determination of size and fluorescence emission of individual BNPs. In this way, the dependence of the fluorescence emission of the commonly used self-inserting lipophilic dye 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine (SP-DiO) could successfully be correlated with nanoparticle size for different types of BNPs, including synthetic lipid vesicles, lipid vesicles derived from cellular membrane extracts, and extracellular vesicles derived from human SH-SY5Y cell cultures; all vesicles had a radius, r, of ∼50 nm and similar size distributions. The results demonstrate that the dependence of fluorescence emission of SP-DiO on nanoparticle size varies significantly between the different types of BNPs, with the expected dependence on membrane area, r2, being observed for synthetic lipid vesicles, while a significant weaker dependence on size was observed for BNPs with more complex composition. The latter observation is attributed to a size-dependent difference in membrane composition, which may influence either the optical properties of the dye and/or the insertion efficiency, indicating that the fluorescence emission of this type of self-inserting dye may not be reliable for determining size or size distribution of BNPs with complex lipid compositions.
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| 7. |
- Lindberg, David, 1986, et al.
(author)
-
Binding of Thioflavin-T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction
- 2017
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 56:16, s. 2170-2174
-
Journal article (peer-reviewed)abstract
- Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addition, thioflavin-T can induce fibril compaction but not alter fibril structure. Our work underscores the photophysical complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quantitative in vitro studies.
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| 8. |
- Midtvedt, Benjamin, et al.
(author)
-
Single-shot self-supervised object detection in microscopy
- 2022
-
In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 13:1
-
Journal article (peer-reviewed)abstract
- Object detection is a fundamental task in digital microscopy, where machine learning has made great strides in overcoming the limitations of classical approaches. The training of state-of-the-art machine-learning methods almost universally relies on vast amounts of labeled experimental data or the ability to numerically simulate realistic datasets. However, experimental data are often challenging to label and cannot be easily reproduced numerically. Here, we propose a deep-learning method, named LodeSTAR (Localization and detection from Symmetries, Translations And Rotations), that learns to detect microscopic objects with sub-pixel accuracy from a single unlabeled experimental image by exploiting the inherent roto-translational symmetries of this task. We demonstrate that LodeSTAR outperforms traditional methods in terms of accuracy, also when analyzing challenging experimental data containing densely packed cells or noisy backgrounds. Furthermore, by exploiting additional symmetries we show that LodeSTAR can measure other properties, e.g., vertical position and polarizability in holographic microscopy.
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| 9. |
- Muslimovic, Aida, et al.
(author)
-
Novel clearance of muscle proteins by muscle cells
- 2020
-
In: European Journal of Cell Biology. - : Elsevier BV. - 0171-9335 .- 1618-1298. ; 99:8
-
Journal article (peer-reviewed)abstract
- Blood levels of cardiac troponins (cTn) and myoglobin are analysed when myocardial infarction (MI) is suspected. Here we describe a novel clearance mechanism for muscle proteins by muscle cells. The complete plasma clearance profile of cTn and myoglobin was followed in rats after intravenous or intermuscular injections and analysed by PET and fluorescence microscopy of muscle biopsies and muscle cells. Compared with intravenous injections, only 5 % of cTnT, 0.6 % of cTnI and 8 % of myoglobin were recovered in the circulation following intramuscular injection. In contrast, 47 % of the renal filtration marker FITC-sinistrin and 81 % of cTn fragments from MI-patients were recovered after intramuscular injection. In addition, PET and biopsy analysis revealed that cTn was taken up by the quadriceps muscle and both cTn and myoglobin were endocytosed by cultured muscle cells. This local clearance mechanism could possibly be the dominant clearance mechanism for cTn, myoglobin and other muscle damage biomarkers released by muscle cells.
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| 10. |
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| 11. |
- Spackova, Barbora, 1979, et al.
(author)
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Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles
- 2022
-
In: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 19, s. 751-758
-
Journal article (peer-reviewed)abstract
- Nanofluidic scattering microscopy enables label-free, quantitative measurements of the molecular weight and hydrodynamic radius of biological molecules and nanoparticles freely diffusing inside a nanofluidic channel. Label-free characterization of single biomolecules aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is technically challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomolecules diffusing inside a nanofluidic channel. NSM facilitates accurate determination of molecular weight from the measured optical contrast and of the hydrodynamic radius from the measured diffusivity, from which information about the conformational state can be inferred. Furthermore, we demonstrate its applicability to the analysis of a complex biofluid, using conditioned cell culture medium containing extracellular vesicles as an example. We foresee the application of NSM to monitor conformational changes, aggregation and interactions of single biomolecules, and to analyze single-cell secretomes.
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| 12. |
- Vilhelmsson Wesén, Emelie, 1989, et al.
(author)
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Role of Membrane Tension Sensitive Endocytosis and Rho GTPases in the Uptake of the Alzheimer's Disease Peptide Aβ(1-42)
- 2020
-
In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:13, s. 1925-1936
-
Journal article (peer-reviewed)abstract
- Intraneuronal accumulation of amyloid-β (Aβ) is an early pathological signum of Alzheimer's disease, and compartments of the endolysosomal system have been implicated in both seeding and cell-cell propagation of Aβ aggregation. We have studied how clathrin-independent mechanisms contribute to Aβ endocytosis, exploring pathways that are sensitive to changes in membrane tension and the regulation of Rho GTPases. Using live cell confocal microscopy and flow cytometry, we show the uptake of monomeric Aβ(1-42) into endocytic vesicles and vacuole-like dilations, following relaxation of osmotic pressure-induced cell membrane tension. This indicates Aβ(1-42) uptake via clathrin independent carriers (CLICs), although overexpression of the bar-domain protein GRAF1, a key regulator of CLICs, had no apparent effect. We furthermore report reduced Aβ(1-42) uptake following overexpression of constitutively active forms of the Rho GTPases Cdc42 and RhoA, whereas modulation of Rac1, which is linked to macropinosome formation, had no effect. Our results confirm that uptake of Aβ(1-42) is clathrin-and dynamin-independent and point to the involvement of a new and distinct clathrin-independent endocytic mechanism which is similar to uptake via CLICs or macropinocytosis but that also appear to involve yet uncharacterized molecular players.
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| 13. |
- Lindberg, David, 1986, et al.
(author)
-
Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by A beta(1-40) and A beta(1-42)
- 2015
-
In: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 458:2, s. 418-423
-
Journal article (peer-reviewed)abstract
- Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the A beta(1-40) and A beta(1-42) variants of the Alzheimer's-related peptide amyloid-beta, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with A beta(1-40) compared to A beta(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with A beta(1-42) fibrils producing a longer ThT fluorescence lifetime compared to A beta(140). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed beta-sheet in the mature A beta fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation.
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| 14. |
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| 15. |
- Muslimovic, Aida, et al.
(author)
-
The Liver and Kidneys mediate clearance of cardiac troponin in the rat
- 2020
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
-
Journal article (peer-reviewed)abstract
- Cardiac-specific troponins (cTn), troponin T (cTnT) and troponin I (cTnI) are diagnostic biomarkers when myocardial infarction is suspected. Despite its clinical importance it is still not known how cTn is cleared once it is released from damaged cardiac cells. The aim of this study was to examine the clearance of cTn in the rat. A cTn preparation from pig heart was labeled with fluorescent dye or fluorine 18 (18 F). The accumulation of the fluorescence signal using organ extracts, or the 18 F signal using positron emission tomography (PET) was examined after a tail vein injection. The endocytosis of fluorescently labeled cTn was studied using a mouse hepatoma cell line. Close to 99% of the cTnT and cTnI measured with clinical immunoassays were cleared from the circulation two hours after a tail vein injection. The fluorescence signal from the fluorescently labeled cTn preparation and the radioactivity from the 18F-labeled cTn preparation mainly accumulated in the liver and kidneys. The fluorescently labeled cTn preparation was efficiently endocytosed by mouse hepatoma cells. In conclusion, we find that the liver and the kidneys are responsible for the clearance of cTn from plasma in the rat.
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| 16. |
- Sasanian, Nima, 1993, et al.
(author)
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Probing physical properties of single amyloid fibrils using nanofluidic channels
- 2023
-
In: European Biophysics Journal. - 1432-1017 .- 0175-7571. ; 52:SUPPL 1, s. S205-S205
-
Conference paper (other academic/artistic)abstract
- Amyloid fibrils formation via protein misfolding and aggregation is associated with many diseases, including neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. The presence of polymorphism phenomenon in a single protein sample makes it important to analyze amyloid fibrils on the single fibril level. We here introduce the concept of nanofluidic channel analysis to the study of single, fluorescently labeled amyloid fibrils. The confinement of amyloid fibrils in nanochannels makes it possible to measure their extension at each width as well as their emission intensity. We used Odijk’s theory for strongly confined polymers to determine the persistence length of each fibril. A majority of the persistence lengths were in the 1-10 m regime and for both the Alzheimer’s protein amyloid- (1-42) and the Parkinson’s protein-synuclein we find at least two populations of fibrils with different persistence lengths, indicating the coexistence of polymorphs with different physical properties. We foresee that the nanofluidics methodology that we have established here will be a useful future tool to study amyloid fibrils on the single fibril level to inform on heterogeneity in their physical properties and interactions.
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| 17. |
- Sasanian, Nima, 1993, et al.
(author)
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Probing physical properties of single amyloid fibrils using nanofluidic channels
- 2023
-
In: Nanoscale. - 2040-3372 .- 2040-3364. ; 15:46, s. 18737-18744
-
Journal article (peer-reviewed)abstract
- Amyloid fibril formation is central to the pathology of many diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Amyloid fibrils can also have functional and scaffolding roles, for example in bacterial biofilms, and have also been exploited as useful biomaterials. Despite being linear protein homopolymers, amyloid fibrils can exhibit significant structural and morphological polymorphism, making it relevant to study them on the single fibril level. We here introduce the concept of nanofluidic channel analysis to the study of single, fluorescently-labeled amyloid fibrils in solution, monitoring the extension and emission intensity of individual fibrils confined in nanochannels with a depth of 300 nm and a width that gradually increases from 300 to 3000 nm. The change in fibril extension with channel width permitted accurate determination of the persistence length of individual fibrils using Odijk's theory for strongly confined polymers. The technique was applied to amyloid fibrils prepared from the Alzheimer's related peptide amyloid-β(1-42) and the Parkinson's related protein α-synuclein, obtaining mean persistence lengths of 5.9 ± 4.5 μm and 3.0 ± 1.6 μm, respectively. The broad distributions of fibril persistence lengths indicate that amyloid fibril polymorphism can manifest in their physical properties. Interestingly, the α-synuclein fibrils had lower persistence lengths than the amyloid-β(1-42) fibrils, despite being thicker. Furthermore, there was no obvious within-sample correlation between the fluorescence emission intensity per unit length of the labelled fibrils and their persistence lengths, suggesting that stiffness may not be proportional to thickness. We foresee that the nanofluidics methodology established here will be a useful tool to study amyloid fibrils on the single fibril level to gain information on heterogeneity in their physical properties and interactions.
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| 18. |
- Wilhelmsson, Marcus, 1974, et al.
(author)
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Meso stereoisomer as a probe of enantioselective threading intercalation of semirigid ruthenium complex [µ-(11,11'-bidppz)(phen)4Ru2]4
- 2003
-
In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 107:42, s. 11784-11793
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Journal article (peer-reviewed)abstract
- Upon interaction with calf thymus DNA the Lambda,Lambda-enantiomer of the semirigid binuclear ruthenium complex [mu-(11, 11'-bidppz)(phen)(4)Ru-2](4+) has previously been shown to reorganize from an initial groove bound geometry into an intercalative binding mode, threading one of its bulky Ru(phen)(2) moieties through the core of the DNA. We have now found that all three stereoisomers, Delta,Delta; Lambda,Lambda; and Delta,Lambda (meso), are intercalated in their final modes of binding to calf thymus DNA, poly(dA-dT)(2), poly(dG-dC)(2), as well as poly(dI-dC)(2) indicated by linear dichroism, circular dichroism, and luminescence. For all three stereoisomers, studied in detail with poly(dA-dT)(2), the bridging bidppz ligand is intercalated in anti conformation, leaving one Ru(phen)(2) Moiety in each groove. This final binding geometry is characterized by a distinct clockwise roll of the Ru(phen)(2) moiety in the minor groove, similar to the roll earlier observed for the dppz ligand in [Ru(phen)(2)dppz](2+). Using the meso stereoisomer as an enantioselective probe, it is shown that the Lambda moiety prefers to insert itself deeply into the minor groove while the Delta moiety, in the major groove, is somewhat displaced from the center of the DNA helix. The preceding, metastable bound geometries are concluded to be in the major groove for calf thymus DNA, poly(dG-dC)(2), and poly(dI-dC)(2), with the Delta,Delta form displaying an angle of the bidppz bridge relative the DNA helix axis of about 50degrees, whereas the corresponding angles for the meso- and Lambda,Lambda-forms in calf thymus DNA are around 65degrees, suggesting an orientation in the groove more parallel to the bases. By contrast, in poly(dA-dT)(2) none of the stereoisomers exhibits any distinguishable initial groove binding mode, but all seem to bind by threading intercalation directly.
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| 19. |
- Zhang, X. A., et al.
(author)
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Dual functions of the human antimicrobial peptide LL-37-Target membrane perturbation and host cell cargo delivery
- 2010
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In: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1798:12, s. 2201-2208
-
Journal article (peer-reviewed)abstract
- The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain ill-defined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced alpha-helix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel alpha-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.
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| 20. |
- Ahlgren, Kajsa, 1996, et al.
(author)
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The inhibition of fibril formation of lysozyme by sucrose and trehalose
- 2024
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In: RSC Advances. - 2046-2069. ; 14:17, s. 11921-11931
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Journal article (peer-reviewed)abstract
- The two disaccharides, trehalose and sucrose, have been compared in many studies due to their structural similarity. Both possess the ability to stabilise and reduce aggregation of proteins. Trehalose has also been shown to inhibit the formation of highly structured protein aggregates called amyloid fibrils. This study aims to compare how the thermal stability of the protein lysozyme at low pH (2.0 and 3.5) is affected by the presence of the two disaccharides. We also address the anti-aggregating properties of the disaccharides and their inhibitory effects on fibril formation. Differential scanning calorimetry confirms that the thermal stability of lysozyme is increased by the presence of trehalose or sucrose. The effect is slightly larger for sucrose. The inhibiting effects on protein aggregation are investigated using small-angle X-ray scattering which shows that the two-component system consisting of lysozyme and water (Lys/H2O) at pH 2.0 contains larger aggregates than the corresponding system at pH 3.5 as well as the sugar containing systems. In addition, the results show that the particle-to-particle distance in the sugar containing systems (Lys/Tre/H2O and Lys/Suc/H2O) at pH 2.0 is longer than at pH 3.5, suggesting larger protein aggregates in the former. Finally, the characteristic distance separating β-strands in amyloid fibrils is observed for the Lys/H2O system at pH 2.0, using wide-angle X-ray scattering, while it is not clearly observed for the sugar containing systems. This study further shows that the two disaccharides stabilise the native fold of lysozyme by increasing the denaturation temperature. However, other factors, such as a weakening of hydrophobic interactions and hydrogen bonding between proteins, might also play a role in their inhibitory effect on amyloid fibril formation.
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| 21. |
- Aliakbarinodehi, Nima, 1986, et al.
(author)
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Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion
- 2022
-
In: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 16:12, s. 20163-20173
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Journal article (peer-reviewed)abstract
- Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.
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| 22. |
- Aran Terol, P., et al.
(author)
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Solvent exposure of Tyr10 as a probe of structural differences between monomeric and aggregated forms of the amyloid-β peptide
- 2015
-
In: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 468:4, s. 696-701
-
Journal article (peer-reviewed)abstract
- Aggregation of amyloid-β (Aβ) peptides is a characteristic pathological feature of Alzheimer's disease. We have exploited the relationship between solvent exposure and intrinsic fluorescence of a single tyrosine residue, Tyr10, in the Aβ sequence to probe structural features of the monomeric, oligomeric and fibrillar forms of the 42-residue Aβ1-42. By monitoring the quenching of Tyr10 fluorescence upon addition of water-soluble acrylamide, we show that in Aβ1-42 oligomers this residue is solvent-exposed to a similar extent to that found in the unfolded monomer. By contrast, Tyr10 is significantly shielded from acrylamide quenching in Aβ1-42 fibrils, consistent with its proximity to the fibrillar cross-β core. Furthermore, circular dichroism measurements reveal that Aβ1-42 oligomers have a considerably lower β-sheet content than the Aβ1-42 fibrils, indicative of a less ordered molecular arrangement in the former. Taken together these findings suggest significant differences in the structural assembly of oligomers and fibrils that are consistent with differences in their biological effects.
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| 23. |
- Baladi, Tom, 1991, et al.
(author)
-
Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery
- 2021
-
In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 143:14, s. 5413-5424
-
Journal article (peer-reviewed)abstract
- Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.
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| 24. |
- Bernson, David, 1986, et al.
(author)
-
Amyloid formation of bovine insulin is retarded in moderately acidic pH and by addition of short-chain alcohols
- 2020
-
In: European Biophysics Journal. - : Springer Science and Business Media LLC. - 1432-1017 .- 0175-7571. ; 49:2, s. 145-153
-
Journal article (peer-reviewed)abstract
- Protein aggregation and amyloid formation are associated with multiple human diseases, but are also a problem in protein production. Understanding how aggregation can be modulated is therefore of importance in both medical and industrial contexts. We have used bovine insulin as a model protein to explore how amyloid formation is affected by buffer pH and by the addition of short-chain alcohols. We find that bovine insulin forms amyloid fibrils, albeit with different rates and resulting fibril morphologies, across a wide pH range (2-7). At pH 4.0, bovine insulin displayed relatively low aggregation propensity in combination with high solubility; this condition was therefore chosen as basis for further exploration of how bovine insulin's native state can be stabilized in the presence of short-chain alcohols that are relevant because of their common use as eluents in industrial-scale chromatography purification. We found that ethanol and isopropanol are efficient modulators of bovine insulin aggregation, providing a three to four times retardation of the aggregation kinetics at 30-35% (vol/vol) concentration; we attribute this to the formation of oligomers, which we detected by AFM. We discuss this effect in terms of reduced solvent polarity and show, by circular dichroism recordings, that a concomitant change in alpha-helical packing of the insulin monomer occurs in ethanol. Our results extend current knowledge of how insulin aggregates, and may, although bovine insulin serves as a simplistic model, provide insights into how buffers and additives can be fine-tuned in industrial production of proteins in general and pharmaceutical insulin in particular.
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| 25. |
- Bost, J. P., et al.
(author)
-
Delivery of Oligonucleotide Therapeutics: Chemical Modifications, Lipid Nanoparticles, and Extracellular Vesicles
- 2021
-
In: Acs Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 15:9, s. 13993-14021
-
Journal article (peer-reviewed)abstract
- Oligonucleotides (ONs) comprise a rapidly growing class of therapeutics. In recent years, the list of FDA-approved ON therapies has rapidly expanded. ONs are small (15-30 bp) nucleotide-based therapeutics which are capable of targeting DNA and RNA as well as other biomolecules. ONs can be subdivided into several classes based on their chemical modifications and on the mechanisms of their target interactions. Historically, the largest hindrance to the widespread usage of ON therapeutics has been their inability to effectively internalize into cells and escape from endosomes to reach their molecular targets in the cytosol or nucleus. While cell uptake has been improved, "endosomal escape" remains a significant problem. There are a range of approaches to overcome this, and in this review, we focus on three: altering the chemical structure of the ONs, formulating synthetic, lipid-based nanoparticles to encapsulate the ONs, or biologically loading the ONs into extracellular vesicles. This review provides a background to the design and mode of action of existing FDA-approved ONs. It presents the most common ON classifications and chemical modifications from a fundamental scientific perspective and provides a roadmap of the cellular uptake pathways by which ONs are trafficked. Finally, this review delves into each of the above-mentioned approaches to ON delivery, highlighting the scientific principles behind each and covering recent advances.
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| 26. |
- Bost, Jeremy P., et al.
(author)
-
Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides
- 2022
-
In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5:1
-
Journal article (peer-reviewed)abstract
- The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.
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| 27. |
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| 28. |
- Esbjörner Winters, Elin, 1978, et al.
(author)
-
Direct Observations of Amyloid beta Self-Assembly in Live Cells Provide Insights into Differences in the Kinetics of A beta(1-40) and A beta(1-42) Aggregation
- 2014
-
In: Chemistry and Biology. - : Elsevier BV. - 1074-5521. ; 21:6, s. 732-742
-
Journal article (peer-reviewed)abstract
- Insight into how amyloid beta (A beta) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of A beta(1-40) and A beta(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of A beta(1-42) aggregation are considerably faster than those of A beta(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how A beta aggregation may occur within neurons.
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| 29. |
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| 30. |
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| 31. |
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| 32. |
- Fant, Kristina, 1979, et al.
(author)
-
DNA condensation by PAMAM dendrimers: Self-assembly characteristics and effect on transcription
- 2008
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 47:6, s. 1732-1740
-
Journal article (peer-reviewed)abstract
- Electrostatic shielding and steric blocking by histones are two significant factors that participate in the control of the local rates of transcription in chromatin. As a simple model system to determine how the degree of DNA condensation affects enzyme accessibility and gene expression, we have used generation 5 polyamidoamine (G5 PAMAM) cationic dendrimer particles (size 5.4 nm) as a synthetic histone model together with an in vitro transcription assay. The degree of compaction, conformation, and binding availability of the dendrimer-DNA complexes is characterized by linear and circular dichroism, dynamic light scattering, and competitive binding of ethidium. Using ultracentrifugation we are able to show explicitly, for the first time, that dendrimer particles bind to DNA in a highly cooperative manner, and that the dendrimer-induced condensation of the DNA strongly attenuates transcription. Two fractions with different properties can be identified: a low-density fraction which behaves very similar to uncondensed DNA and a high-density fraction which is condensed to a high extent and where binding availability and transcription are strongly reduced. Circular dichroism gives clues to the structure of the condensed DNA indicating long-range order between the helices such as in polymer-salt-induced cholesteric liquid crystalline domains, one possible shape being a toroidal structure. On the basis of the experimental data, we propose a model for the self-assembly of the dendrimer-DNA system.
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| 33. |
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| 34. |
- Ghaeidamini, Marziyeh, 1989, et al.
(author)
-
Graphene oxide sheets and quantum dots inhibit alpha-synuclein amyloid formation by different mechanisms
- 2020
-
In: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 12:37, s. 19450-19460
-
Journal article (peer-reviewed)abstract
- Aggregation and amyloid formation of the 140-residue presynaptic and intrinsically disordered protein alpha-synuclein (alpha-syn) is a pathological hallmark of Parkinson's disease (PD). Understanding how alpha-syn forms amyloid fibrils, and investigations of agents that can prevent their formation is therefore important. We demonstrate herein that two types of graphene oxide nanoparticles (sheets and quantum dots) inhibit alpha-syn amyloid formation by different mechanisms mediatedviadifferential interactions with both monomers and fibrils. We have used thioflavin-T fluorescence assays and kinetic analysis, circular dichroism, dynamic light scattering, fluorescence spectroscopy and atomic force microscopy to asses the kinetic nature and efficiency of this inhibitory effect. We show that the two types of graphene oxide nanoparticles alter the morphology of alpha-syn fibrils, disrupting their interfilament assembly and the resulting aggregates therefore consist of single protofilaments. Our results further show that graphene oxide sheets reduce the aggregation rate of alpha-syn primarily by sequestering of monomers, thereby preventing primary nucleation and elongation. Graphene quantum dots, on the other hand, interact less avidly with both monomers and fibrils. Their aggregation inhibitory effect is primarily related to adsorption of aggregated species and reduction of secondary processes, and they can thus not fully prevent aggregation. This fine-tuned and differential effect of graphene nanoparticles on amyloid formation shows that rational design of these nanomaterials has great potential in engineering materials that interact with specific molecular events in the amyloid fibril formation process. The findings also provide new insight into the molecular interplay between amyloidogenic proteins and graphene-based nanomaterials in general, and opens up their potential use as agents to manipulate fibril formation.
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| 35. |
- Hagelin, Christina, 1977, et al.
(author)
-
Assigning Membrane Binding Geometry of Cytochrome c by Polarized Light Spectroscopy
- 2009
-
In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 96:8, s. 3399-3411
-
Journal article (peer-reviewed)abstract
- In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) analysis of the peptide ultraviolet-visible spectrum of a membrane-associated protein (cytochrome (cyt) c) allows orientation and structure to be assessed with quite high accuracy in a native membrane environment that can be systematically varied with respect to lipid composition. Cyt c binds strongly to negatively charged lipid bilayers with a distinct orientation in which its a-helical segments are on average parallel to the membrane surface. Further information is provided by the LID of the pi-pi* transitions of the heme porphyrin and transitions of aromatic residues, mainly a single tryptophan. A good correlation with NMR data was found, and combining NMR structural data with LID angular data allowed the whole protein to be docked to the lipid membrane. When the redox state of cyt c was changed, distinct variations in the LID spectrum of the heme Soret band were seen corresponding to changes in electronic transition energies; however, no significant change in the overall protein orientation or structure was observed. Cyt c is known to interact in a specific manner with the doubly negatively charged lipid cardiolipin, and incorporation of this lipid into the membrane at physiologically relevant levels was indeed found to affect the protein orientation and its a-helical content. The detail in which cyt c binding is described in this study shows the potential of LID spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.
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| 36. |
- Hagelin, Christina, 1977, et al.
(author)
-
Membrane interactions of cell-penetrating peptides probed by tryptophan fluorescence and dichroism techniques: Correlations of structure to cellular uptake
- 2006
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 45:24, s. 7682-7692
-
Journal article (peer-reviewed)abstract
- This work reports on the binding and conformation of a series of CPPs in the bilayer membranes of large unilamellar vesicles and the effect of the presence of cholesterol. We show a negative correlation between alpha-helical structure and uptake efficiency for penetratin peptides where the two central arginine residues of penetratin are thought to be important for breaking the secondary structure. Penetratin alpha-helicity is also reduced upon incorporation of cholesterol into the membrane. Flow linear dichroism in the far-UV region shows that the penetratin peptides adopt a preferential orientation of the alpha-helix parallel to the bilayer, and the linear dichroism (LD) spectrum in the aromatic region indicates that the tryptophan residues are preferentially oriented parallel to the membrane. The Tat analogue TatP59W and the oligoarginine R7W, which are more efficient CPPs than penetratin, bind to membranes as random coils and do not show any orientation in LD, again indicating that alpha-helicity reduces uptake efficiency. Further, we observe large variations in tryptophan quantum yields for the five CPPs in this study and discuss this in terms of the ability to cause lipid rearrangement. Binding isotherms show that cholesterol increases the affinity of the peptide for the membrane, but tryptophan fluorescence lifetimes are essentially unaltered by incorporation of as much as 40 mol % cholesterol into the membrane, suggesting the absence of specific peptide-cholesterol interactions. Fluorescence emission maxima are insensitive to cholesterol and indicate that the peptide is positioned in the headgroup region. The results on peptide-membrane interactions are discussed in terms of possible uptake mechanisms.
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| 37. |
- Havemeister, Fritjof, 1994, et al.
(author)
-
Monovalent cations have different effects on the assembly kinetics and morphology of α-synuclein amyloid fibrils
- 2023
-
In: Biochemical and Biophysical Research Communications. - 1090-2104 .- 0006-291X. ; 679, s. 31-36
-
Journal article (peer-reviewed)abstract
- Formation of α-synuclein amyloid fibrils is a pathological hallmark of Parkinson's disease and a phenomenon that is strongly modulated by environmental factors. Here, we compared effects of different monovalent cations (Li+, Na+, K+) on the formation and properties of α-synuclein amyloid fibrils. Na+ > Li+ were found to have concentration-dependent catalytic effects on primary nucleation whereas K+ ions acted inhibitory. We discuss this discrepancy in terms of a superior affinity of Na+ and Li+ to carboxylic protein groups, resulting in reduced Columbic repulsion and by considering K+ as an ion with poor protein binding and slight chaotropic character, which could promote random coil protein structure. K+ ions, furthermore, appeared to lower the β-sheet content of the fibrils and increase their persistence lengths, the latter we interpret as a consequence of lesser ion binding and hence higher line charge of the fibrils. The finding that Na+ and K+ have opposite effects on α-synuclein aggregation is intriguing in relation to the significant transient gradients of these ions across axonal membranes, but also important for the design and interpretation of biophysical assays where buffers containing these monovalent cations have been intermixedly used.
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| 38. |
- Lindberg, David, 1986, et al.
(author)
-
Detection of amyloid-beta fibrils using the DNA-intercalating dye YOYO-1: Binding mode and fibril formation kinetics
- 2016
-
In: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 469:2, s. 313-318
-
Journal article (peer-reviewed)abstract
- Identification of the chemical and biological properties of amyloid fibrils is important for understanding their roles in human diseases and to clarify the mechanisms that govern their formation. In pursuit of these goals, small molecule fluorescent dyes have received increasing attention as probes of amyloid conformations. In this study, we report on the ability of YOYO-1, a homodimeric derivative of oxazole yellow, to detect fibrils formed by the Alzheimer's disease related A beta(1-42) peptide. We find that YOYO-1 binds to A beta(1-42) fibrils with the long axes of its oxazole yellow moieties parallel to the fibril axis, resulting in a 200x emission enhancement; a result that shows that YOYO-1 is a sensitive amyloid probe. Further, YOYO-1 exhibits characteristic absorption shifts upon binding to the A beta(1-42) fibrils that we attribute to a self-stacking to non-stacking transition in its homodimer configuration; herein we show how this phenomenon can be exploited to estimate the degree of dye binding. Furthermore, we show that YOYO-1 can be used to monitor the kinetics of amyloid formation reactions. Taken together, our results show that YOYO-1 is a sensitive amyloid probe that can operate with both absorption and fluorescence read-outs, and this suggests that this commercially available dye could become a useful complement to thioflavin-T for in vitro amyloid-sensing applications.
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| 39. |
- Lindberg, David, 1986, et al.
(author)
-
Lipid membranes catalyse the fibril formation of the amyloid-? (1–42) peptide through lipid-fibril interactions that reinforce secondary pathways
- 2017
-
In: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1859:10, s. 1921-1929
-
Journal article (peer-reviewed)abstract
- Alzheimer's disease is associated with the aggregation of amyloid-? (A?) peptides into oligomers and fibrils. We have explored how model lipid membranes modulate the rate and mechanisms of A?(1–42) self-assembly, in order to shed light on how this pathological reaction may occur in the lipid-rich environments that the peptide encounters in the brain. Using a combination of in vitro biophysical experiments and theoretical approaches, we show that zwitterionic DOPC lipid vesicles accelerate the A?(1–42) fibril growth rate by interacting specifically with the growing fibrils. We probe this interaction with help of a purpose-developed Förster resonance energy transfer assay that monitors the proximity between a fibril-specific dye and fluorescent lipids in the lipid vesicle membrane. To further rationalise these findings we use mathematical models to fit the aggregation kinetics of A?(1–42) and find that lipid vesicles alter specific mechanistic steps in the aggregation reaction; they augment monomer-dependent secondary nucleation at the surface of existing fibrils and facilitate monomer-independent catalytic processes consistent with fibril fragmentation. We further show that DOPC vesicles have no effect on primary nucleation. This finding is consistent with experiments showing that A?(1–42) monomers do not directly bind to the lipid bilayer. Taken together, our results show that plain lipid membranes with charge and composition that is representative of outer cell membranes can significantly augment autocatalytic steps in the self-assembly of A?(1–42) into fibrils. This new insight suggests that strategies to reduce fibril-lipid interactions in the brain may have therapeutic value.
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| 40. |
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| 41. |
- Matson Dzebo, Maria, 1985, et al.
(author)
-
Copper Chaperone Atox1 Interacts with Cell Cycle Proteins
- 2018
-
In: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 16, s. 443-449
-
Journal article (peer-reviewed)abstract
- The anaphase-promoting complex (APC) is involved in several processes in the cell cycle, most prominently it facilitates the separation of the sister chromatids during mitosis, before cell division. Because of the key role in the cell cycle, APC is suggested as a putative target for anticancer agents. We here show that the copper chaperone Atox1, known for shuttling copper in the cytoplasm from Ctr1 to ATP7A/B in the secretory pathway, interacts with several APC subunits. Atox1 interactions with APC subunits were discovered by mass spectrometry of co-immunoprecipitated samples and further confirmed using proximity ligation assays in HEK293T cells. Upon comparing wild-type cells with those in which the Atox1 gene had been knocked out, we found that in the absence of Atox1 protein, cells have prolonged G2/M phases and a slower proliferation rate. Thus, in addition to copper transport for loading of copper-dependent enzymes, Atox1 may modulate the cell cycle by interacting with APC subunits.
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| 42. |
- Munson, Michael J., et al.
(author)
-
A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery
- 2021
-
In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 4:1
-
Journal article (peer-reviewed)abstract
- RNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Development and optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by limited screening methods to probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. Furthermore, this assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We identify nanoparticles with superior escape properties and demonstrate cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.
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| 43. |
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| 44. |
- Ng, Janice S.W., et al.
(author)
-
Using Tetracysteine-Tagged TDP-43 with a Biarsenical Dye to Monitor Real-Time Trafficking in a Cell Model of Amyotrophic Lateral Sclerosis
- 2019
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 58:39, s. 4086-4095
-
Journal article (peer-reviewed)abstract
- TAR DNA-binding protein 43 (TDP-43) has been identified as the major constituent of the proteinaceous inclusions that are characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin positive frontotemporal lobar degeneration (FTLD). Wild type TDP-43 inclusions are a pathological hallmark of >95% of patients with sporadic ALS and of the majority of familial ALS cases, and they are also found in a significant proportion of FTLD cases. ALS is the most common form of motor neuron disease, characterized by progressive weakness and muscular wasting, and typically leads to death within a few years of diagnosis. To determine how the translocation and misfolding of TDP-43 contribute to ALS pathogenicity, it is crucial to define the dynamic behavior of this protein within the cellular environment. It is therefore necessary to develop cell models that allow the location of the protein to be defined. We report the use of TDP-43 with a tetracysteine tag for visualization using fluorogenic biarsenical compounds and show that this model displays features of ALS observed in other cell models. We also demonstrate that this labeling procedure enables live-cell imaging of the translocation of the protein from the nucleus into the cytosol.
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| 45. |
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| 46. |
- Nilsson, Jesper, 1984, et al.
(author)
-
Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics
- 2021
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 11:1
-
Journal article (peer-reviewed)abstract
- To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we herein explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. Both cytosine and adenine analogues (tC, tCO, 2CNqA, and pA) were incorporated into a 16mer ASO sequence with a 3-10-3 cEt-DNA-cEt (cEt = constrained ethyl) gapmer design. In addition to a comprehensive photophysical characterization, we assess the label-induced effects on the gapmers’ RNA affinities, RNA-hybridized secondary structures, and knockdown efficiencies. Importantly, we find practically no perturbing effects for gapmers with single FBA incorporations in the biologically critical gap region and, except for pA, the FBAs do not affect the knockdown efficiencies. Incorporating two cytosine FBAs in the gap is equally well tolerated, while two adenine analogues give rise to slightly reduced knockdown efficiencies and what could be perturbed secondary structures. We furthermore show that the FBAs can be used to visualize gapmers inside live cells using fluorescence microscopy and flow cytometry, enabling comparative assessment of their uptake. This altogether shows that FBAs are functional ASO probes that provide a minimally perturbing in-sequence labeling option for this highly relevant drug modality.
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| 47. |
- Persson, Daniel, 1972, et al.
(author)
-
Vesicle size-dependent translocation of penetratin analogs across lipid membranes
- 2004
-
In: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1665:1-2, s. 142-155
-
Journal article (peer-reviewed)abstract
- The recent discoveries of serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides (CPPs) have prompted a reevaluation of the current understanding of peptide-mediated cellular delivery. Following a report on the differential cellular uptake of a number of penetratin analogs in unfixed cells, we here investigate their membrane translocation abilities in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Surprisingly, in contrast to the behavior in living cells, all peptides readily entered the giant vesicles ( > 1 μm) as proved by confocal microscopy, while none of them could cross the membranes of LUVs (100 nm). For determination of the location of the peptides in the LUVs, a new concept was introduced, based on sensitive resonance energy transfer (RET) measurements of the enhanced fluorescence of acceptor fluorophores present solely in the inner leaflet. An easily adopted method to prepare such asymmetrically labeled liposomes is described. The membrane insertion depths of the tryptophan moieties of the peptides were determined by use of brominated lipids and found to be very similar for all of the peptides studied. We also demonstrate that infrared spectroscopy on the lipid carbonyl stretch vibration peak is a convenient technique to determine phospholipid concentration. © 2004 Elsevier B.V. All rights reserved.
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| 48. |
- Rahimi, Shadi, 1982, et al.
(author)
-
Ginsenoside Rg3 Reduces the Toxicity of Graphene Oxide Used for pH-Responsive Delivery of Doxorubicin to Liver and Breast Cancer Cells
- 2023
-
In: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 15:2
-
Journal article (peer-reviewed)abstract
- Doxorubicin (DOX) is extensively used in chemotherapy, but it has serious side effects and is inefficient against some cancers, e.g., hepatocarcinoma. To ameliorate the delivery of DOX and reduce its side effects, we designed a pH-responsive delivery system based on graphene oxide (GO) that is capable of a targeted drug release in the acidic tumor microenvironment. GO itself disrupted glutathione biosynthesis and induced reactive oxygen species (ROS) accumulation in human cells. It induced IL17-directed JAK-STAT signaling and VEGF gene expression, leading to increased cell proliferation as an unwanted effect. To counter this, GO was conjugated with the antioxidant, ginsenoside Rg3, prior to loading with DOX. The conjugation of Rg3 to GO significantly reduced the toxicity of the GO carrier by abolishing ROS production. Furthermore, treatment of cells with GO–Rg3 did not induce IL17-directed JAK-STAT signaling and VEGF gene expression—nor cell proliferation—suggesting GO–Rg3 as a promising drug carrier. The anticancer activity of GO–Rg3–DOX conjugates was investigated against Huh7 hepatocarcinoma and MDA-MB-231 breast cancer cells. GO–Rg3–DOX conjugates significantly reduced cancer cell viability, primarily via downregulation of transcription regulatory genes and upregulation of apoptosis genes. GO–Rg3 is an effective, biocompatible, and pH responsive DOX carrier with potential to improve chemotherapy—at least against liver and breast cancers.
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| 49. |
- Rydberg, Hanna, 1982, et al.
(author)
-
Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides
- 2012
-
In: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 51:27, s. 5531-5539
-
Journal article (peer-reviewed)abstract
- Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view.
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| 50. |
- Sasanian, Nima, 1993, et al.
(author)
-
Redox-Dependent Copper Ion Modulation of Amyloid-β (1-42) Aggregation In Vitro
- 2020
-
In: Biomolecules. - : MDPI AG. - 2218-273X. ; 10:6, s. 1-19
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Journal article (peer-reviewed)abstract
- Plaque deposits composed of amyloid-β (Aβ) fibrils are pathological hallmarks of Alzheimer’s disease (AD). Although copper ion dyshomeostasis is apparent in AD brains and copper ions are found co-deposited with Aβ peptides in patients’ plaques, the molecular effects of copper ion interactions and redox-state dependence on Aβ aggregation remain elusive. By combining biophysical and theoretical approaches, we here show that Cu2+ (oxidized) and Cu+ (reduced) ions have opposite effects on the assembly kinetics of recombinant Aβ(1-42) into amyloid fibrils in vitro. Cu2+ inhibits both the unseeded and seeded aggregation of Aβ(1-42) at pH 8.0. Using mathematical models to fit the kinetic data, we find that Cu2+ prevents fibril elongation. The Cu2+-mediated inhibition of Aβ aggregation shows the largest effect around pH 6.0 but is lost at pH 5.0, which corresponds to the pH in lysosomes. In contrast to Cu2+, Cu+ ion binding mildly catalyzes the Aβ(1-42) aggregation via a mechanism that accelerates primary nucleation, possibly via the formation of Cu+-bridged Aβ(1-42) dimers. Taken together, our study emphasizes redox-dependent copper ion effects on Aβ(1-42) aggregation and thereby provides further knowledge of putative copper-dependent mechanisms resulting in AD.
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