| 1. |
- Leong, Swee, et al.
(författare)
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A real world pilot implementation of the Core Manufacturing Simulation Data model
- 2008
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Ingår i: ; , s. 334-341
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Konferensbidrag (refereegranskat)abstract
- While software for discrete event simulation (DES) has emerged into sophisticated tools for decision support in a wide range of contexts, the need to integrate DES tools with other applications is increasing. In the industrial engineering context, simulation engineers strive to use real world data, e.g., logs of machine breakdown, to make behavior of DES models imitate reality. However, the format used for describing simulation data is often specialized to the current situation. The Core Manufacturing Simulation Data (CMSD) is a collaborative effort with academia and industry to standardize the format used for simulation data, to facilitate data exchange among simulation and manufacturing applications. This paper describes the results from a pilot implementation study at Volvo Trucks, where CMSD was utilized as the data exchange format between two data systems and two DES models. The DES tools used were commercial software packages Unigraphics Plant Simulation and InControl Enterprise Dynamics. Generic and reusable interfaces for CMSD-file communication were developed for each of these tools. The CMSD interfaces were successfully connected to a model in each simulation tool describing the same manufacturing process. A stand alone application was developed to collect and analyze raw data and to create the CMSD file being used as input data for both models. The result is a system including raw data analysis, data reformatting, CMSD interfacing, and model execution. Based on the result, a generic methodology for CMSD interface development in DES tools has evolved. The most important conclusion is that CMSD data can be interpretable by both Plant Simulation and Enterprise Dynamics, and that it saves engineering development time during the model building phase.
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| 2. |
- Lundgren, Anders, 1978, et al.
(författare)
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Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations
- 2018
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 18:1, s. 381-385
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Tidskriftsartikel (refereegranskat)abstract
- The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analyzed for their function with single-molecule microscopy. Expansion of this approach toward a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilization and reconstitution often cause protein conformational changes and loss of membrane-specific cofactors, which may impair protein function. To overcome this limitation, we here demonstrate how antibody-modified nanoparticles can be used to achieve affinity purification and enrichment of selected integral membrane proteins directly from cell membrane preparations. Nanoparticles were first bound to the ectodomain of β-secretase 1 (BACE1) contained in cell-derived membrane vesicles. In a subsequent step, these were merged into a continuous supported membrane in a microfluidic channel. Through the extended nanoparticle tag, a weak (∼fN) hydrodynamic force could be applied, inducing directed in-membrane movement of targeted BACE1 exclusively. This enabled selective thousand-fold enrichment of the targeted membrane protein while preserving a natural lipid environment. In addition, nanoparticle-targeting also enabled simultaneous tracking analysis of each individual manipulated protein, revealing how their mobility changed when moved from one lipid environment to another. We therefore believe this approach will be particularly useful for separation in-line with single-molecule analysis, eventually opening up for membrane-protein sorting devices analogous to fluorescence-activated cell sorting.
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| 3. |
- Block, Stephan, 1978, et al.
(författare)
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Two-dimensional flow nanometry of biological nanoparticles for accurate determination of their size and emission intensity
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 7, s. art no 12956 -
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Tidskriftsartikel (refereegranskat)abstract
- Biological nanoparticles (BNPs) are of high interest due to their key role in various biological processes and use as biomarkers. BNP size and composition are decisive for their functions, but simultaneous determination of both properties with high accuracy remains challenging. Optical microscopy allows precise determination of fluorescence/scattering intensity, but not the size of individual BNPs. The latter is better determined by tracking their random motion in bulk, but the limited illumination volume for tracking this motion impedes reliable intensity determination. Here, we show that by attaching BNPs to a supported lipid bilayer, subjecting them to hydrodynamic flows and tracking their motion via surface-sensitive optical imaging enable determination of their diffusion coefficients and flow-induced drifts, from which accurate quantification of both BNP size and emission intensity can be made. For vesicles, the accuracy of this approach is demonstrated by resolving the expected radius-squared dependence of their fluorescence intensity for radii down to 15 nm.
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| 4. |
- Carlred, Louise M, 1985, et al.
(författare)
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Imaging of Amyloid-β in Alzheimer’s disease transgenic mouse brains with Time-of-Flight Secondary Ion Mass Spectrometry using Immunoliposomes
- 2016
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Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 11:2, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-β (Aβ), a biologically relevant peptide in Alzheimer's disease (AD), in transgenic mouse braintissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aβ. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aβ deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid–protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.
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| 5. |
- Carlred, Louise M, 1985, et al.
(författare)
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Simultaneous imaging of amyloid-β and lipids in brain tissue using antibody-coupled liposomes and time-of-flight secondary ion mass spectrometry
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 136:28, s. 9973-9981
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Tidskriftsartikel (refereegranskat)abstract
- The spatial localization of amyloid-β peptide deposits, the major component of senile plaques in Alzheimer's disease (AD), was mapped in transgenic AD mouse brains using time-of-flight secondary ion mass spectrometry (ToF-SIMS), simultaneously with several endogenous molecules that cannot be mapped using conventional immunohistochemistry imaging, including phospholipids, cholesterol and sulfatides. Whereas the endogenous lipids were detected directly, the amyloid-β deposits, which cannot be detected as intact entities with ToF-SIMS because of extensive ion-induced fragmentation, were identified by specific binding of deuterated liposomes to antibodies directed against amyloid-β. Comparative investigation of the amyloid-β deposits using conventional immunohistochemistry and fluorescence microscopy suggests similar sensitivity but a more surface-confined identification due to the shallow penetration depth of the ToF-SIMS signal. The recorded ToF-SIMS images thus display the localization of lipids and amyloid-β in a narrow (∼10 nm) two-dimensional plane at the tissue surface. As compared to a frozen nontreated tissue sample, the liposome preparation protocol generally increased the signal intensity of endogenous lipids, likely caused by matrix effects associated with the removal of salts, but no severe effects on the tissue integrity and the spatial distribution of lipids were observed with ToF-SIMS or scanning electron microscopy (SEM). This method may provide an important extension to conventional tissue imaging techniques to investigate the complex interplay of different kinds of molecules in neurodegenerative diseases, in the same specimen. However, limitations in target accessibility of the liposomes as well as unspecific binding need further consideration. © 2014 American Chemical Society.
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| 6. |
- Johansson Fast, Björn, 1986
(författare)
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Applications of hydrodynamic forces for membrane chromatography
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins are proteins that reside in the cell membrane. Due to their position in the cell membrane, the barrier between the cell interior and exterior, they are vital to cell signaling. Moreover, the presence of membrane proteins in the cell's signaling pathways make membrane proteins important drug targets. In fact, one of the most important drug target classes are the membrane residing G protein coupled receptors. Despite this apparent importance there is currently no good technique for purifying membrane proteins that does not also have a high risk of causing denaturation of the protein. Taking into account that the cell membrane and its constituents can be described as a two-dimensional fluid, combined with the fact that many chromatography techniques are two-dimensional systems, it may seem peculiar that membrane chromatography is not yet solved in a satisfactory way. The aim of this thesis is to contribute to improved membrane protein chromatography.Hydrodynamic forces have recently been used to induce lateral movement of membrane associated molecules, a principle which has been utilized to improve the efficiency of the accumulation and separation of membrane associated molecules situated in supported lipid bilayers (SLB). This was done by forming SLBs in microfluidic channels, and by further introducing a gold barrier situated on the floor of the microfluidic channel. The barrier restricts the SLB to the center of the microfluidic channel where the spatial variation of the hydrodynamic force is small. Thanks to the so obtained homogeneous force, it was possible to show that separation of cholera toxin B subunit based on the number of attachment points to the SLB was complete, with no material in between the different populations.Also, a method for label-free diffusivity measurements on a substrate that is sensitive to changes in the effective refractive index in a small volume above the substrate is described. The technique is based on local application of hydrodynamic forces, in a so called hydrodynamic trap, to locally accumulate proteins at a surface coverage significantly higher than the equilibrium of the system. The trap is subsequently turned off, allowing the accumulated proteins to diffuse out of the trap, allowing the diffusivity of the accumulated proteins can be studied. The latter method was shown to provide a unique possibility to compare the diffusivity of labeled and non-labeled proteins in otherwise identical systems. It was shown that the inclusion of fluorescent labels decreases the diffusivity by approximately 12%.
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| 7. |
- Johansson Fast, Björn, 1986
(författare)
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Hydrodynamic flow for deterministic sorting of cell-membrane components
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The biological membrane, an amphiphilic structure that is the barrier between the cell interior and exterior, is one of the vital building blocks of all cells. Not only does it define the outer boundaries of the cell, it also carries important biological function by virtue of the proteins and other molecules that constitute the membrane, the function of which is in turn intimately coupled to their association with the lipid-based membrane.That the function of the protein is linked to its amphiphilicity necessitates preservation of the amphiphilic environment when probing the function of membrane associated proteins. The low expression levels of, even overexpressed, membrane proteins in a cell membrane crowded with many different types of proteins presents a barrier for direct studies of this class of proteins in their native membrane environment.In the work leading up to this thesis, the aim has been to overcome some of these hurdles and enable membrane protein accumulation and purification in a near native cell membrane. In the articles appended to the thesis, steps have been taken towards being able to move, concentrate, purify and in the end visualize single membrane proteins, using a combination of surface-sensitive imaging, microfluidics and hydrodynamic flow. The last development led to the insight that using this approach we were able to determine the exact size of nanometer-sized objects bound to the two-dimensional interface that is the supported membrane by measuring both the nanoparticles' deterministic and stochastic movement.Looking forward, this thesis work has provided a solid foundation for deterministic sorting of membrane proteins, without the need for detergent solubilization. While this in itself is rather enticing, the possibility to simultaneously determine both the size and the biomolecular content of biological nanoparticles, as demonstrated in the final paper, might help telling whether it is the size, the amount of a specific molecule, or a precise combination of the two, that is decisive for their biological function, such as; infectivity, gene transfer or drug delivery in the context of virions, exosomes and nanoscale drug carriers, respectively.
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| 8. |
- Johansson Fast, Björn, 1986, et al.
(författare)
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Hydrodynamic separation of proteins in supported lipid bilayers confined by gold barriers
- 2013
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 9:39, s. 9414-9419
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Tidskriftsartikel (refereegranskat)abstract
- Hydrodynamic drag forces generated by liquid flow above a supported lipid bilayer (SLB) can be used to induce lateral movement of molecules protruding from the SLB. Since the velocity of the individual molecules depends on their size and coupling to the lipid bilayer, these forces can also be used to enrich and separate different types of membrane-bound molecules. To improve and better quantify hydrodynamic-based molecular separation in SLBs, we formed the SLB on the floor of a microfluidic channel which was patterned with gold barriers that confined the lipid bilayer to a 100 mm wide strip in the center of a 300 mm wide microfluidic channel. This forces the SLB into a region of the channel where the spatial variation of the hydrodynamic forces is close to zero while at the same time preventing the SLB from creeping up on the PDMS sides of the channel, thus reducing the loss of material. We here use this approach to investigate the accumulation of (i) fluorescently labeled lipids and (ii) the protein complex cholera toxin B (CTB) and to compare how the accumulation and separation differ when having an infinite reservoir or only a spatially limited band of studied molecules in the SLB. In addition, we show how the method can be used for complete separation of different polyvalently bound fractions of CTB.
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| 9. |
- Johansson Fast, Björn, 1986, et al.
(författare)
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Label-Free Measurements of the Diffusivity of Molecules in Lipid Membranes
- 2014
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Ingår i: ChemPhysChem. - : Wiley. - 1439-7641 .- 1439-4235. ; 15:3, s. 486-491
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Tidskriftsartikel (refereegranskat)abstract
- An important and characteristic property of a cell membrane is the lateral mobility of protein molecules in the lipid bilayer. This has conventionally been measured by labeling the molecules with fluorescent markers and monitoring their mobility by different fluorescence-based techniques. However, adding the label to the studied molecule may affect the system, so it is an assumption in almost all experiments that the measured mobility of the biomolecule with its label is the same as that of the unlabeled molecule. However, this assumption is rarely tested due to a lack of suitable methods. In this work, a new technique to perform label-free diffusivity measurements is developed and used to measure the effect of the label for two common protein-lipid systems: 1) streptavidin (SA) coupled to a supported lipid bilayer (SLB) through biotinylated lipids and 2) the extracellular part of the T-cell adhesion protein CD2, coupled to an SLB through histidine tags to nickel-chelating lipids. A measurable (approximate to 12%) decrease in diffusivity is found for both labeled proteins, even though the molecular mass of the label is almost 100 times smaller than those of the proteins (approximate to 50 kDa). The results illustrate the importance of being able to study different biophysical properties of cell membranes and their mimics without relying on fluorescent labels, especially if fluorescent labeling is difficult or is expected to affect the nature of the intermolecular interactions being studied.
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