| 1. |
- Bonander, Nicklas, 1968, et al.
(författare)
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Optimising yeast as a host for recombinant protein production (review)
- 2012
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029 .- 1064-3745. ; 866, s. 1-9
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Forskningsöversikt (refereegranskat)abstract
- Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.
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| 2. |
- Campbell, Kate, 1987, et al.
(författare)
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Self-Establishing Communities: A Yeast Model to Study the Physiological Impact of Metabolic Cooperation in Eukaryotic Cells
- 2019
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; , s. 263-282
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- All biosynthetically active cells are able to export and import metabolites, the small molecule intermediaries of metabolism. In dense cell populations, this hallmark of cells results in the intercellular exchange of a wide spectrum of metabolites. Such metabolite exchange enables metabolic specialization of individual cells, leading to far reaching biological implications, as a consequence of the intrinsic connection between metabolism and cell physiology. In this chapter, we discuss methods on how to study metabolite exchange interactions by using self-establishing metabolically cooperating communities (SeMeCos) in the budding yeast Saccharomyces cerevisiae. SeMeCos exploit the stochastic segregation of episomes to progressively increase the number of essential metabolic interdependencies in a community that grows out from an initially prototrophic cell. By coupling genotype to metabotype, SeMeCos allow for the tracking of cells while they specialize metabolically and hence the opportunity to study their progressive change in physiology.
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| 3. |
- Chen, Yu, 1990, et al.
(författare)
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Genome-Scale Metabolic Modeling from Yeast to Human Cell Models of Complex Diseases: Latest Advances and Challenges
- 2019
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; , s. 329-345
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Genome-scale metabolic models (GEMs) are mathematical models that enable systematic analysis of metabolism. This modeling concept has been applied to study the metabolism of many organisms including the eukaryal model organism, the yeast Saccharomyces cerevisiae, that also serves as an important cell factory for production of fuels and chemicals. With the application of yeast GEMs, our knowledge of metabolism is increasing. Therefore, GEMs have also been used for modeling human cells to study metabolic diseases. Here we introduce the concept of GEMs and provide a protocol for reconstructing GEMs. Besides, we show the historic development of yeast GEMs and their applications. Also, we review human GEMs as well as their uses in the studies of complex diseases.
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| 4. |
- Davidsson, Johan, 1967, et al.
(författare)
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Experimental Models for Neurotrauma Research
- 2016
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Ingår i: Methods in Molecular Biology. - 1940-6029 .- 1064-3745. ; 1462, s. 267-88
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Tidskriftsartikel (refereegranskat)abstract
- Physical trauma in the central nervous system (CNS) is usually the result of a number of forces in different directions and dimensions. A large number of experimental models have been developed to improve the possibilities to understand the outcome of CNS trauma. In this chapter, we will describe the need for a variety of experimental models for research on traumatic brain injury (TBI) and spinal cord injury (SCI). Models can serve different needs, such as: to test new treatments for injuries, to reveal thresholds for injuries, to provide a better understanding of injury mechanisms, or to test tools and methods for translation between experiments and clinical data. In this chapter, we will discuss on the validation of models and translation between experimental and clinical studies.
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| 5. |
- Engqvist, Martin, 1983, et al.
(författare)
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Metabolic engineering of photorespiration
- 2017
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; 1653, s. 137-155
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The introduction of two alternative glycolate catabolic pathways in the chloroplasts of Arabidopsis thaliana rendered plants with increased biomass. To introduce these synthetic pathways, the selected genes were stepwise integrated in the nuclear genome of wild-type plants. These plants were transformed by Agrobacterium tumefaciens carrying the binary vectors using the floral dip method. Selection of transformants was conducted using different selection agents and the expression of the transgenes was confirmed by PCR and enzyme activity measurements.
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| 6. |
- Frost, Rickard, 1979, et al.
(författare)
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Characterization of nanoparticle-lipid membrane interactions using QCM-D
- 2013
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029 .- 1064-3745. - 9781627033350 ; 991, s. 127-137
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- In vitro characterization of nanoparticles is becoming increasingly important due to the rapid development of novel nanoparticle formulations for applications in the field of nanomedicine and related areas. Commonly, nanoparticles are simply characterized with respect to their size and zeta potential, and additional in vitro characterization of nanoparticles is needed to develop useful nanoparticle structure-activity relationships. In this context it is highly interesting to characterize the interactions between nanoparticles and model interfaces, such as lipid membranes. Here, we describe a methodology to study such interactions using the quartz crystal microbalance with dissipation monitoring technique (QCM-D). In order to mimic some aspects of the native cell membrane, a supported lipid membrane is formed on the QCM-D sensor surface. Subsequently the membrane is exposed to nanoparticles, and the nanoparticle-lipid membrane interactions are monitored in real time. The outcome of such analysis provides information on the adsorption process (importantly kinetics and adsorbed amounts) as well as on the integrity of both the nanoparticles and the lipid membrane upon interaction. QCM-D analyses are suitable for screening of nanoparticle-lipid membrane interactions due to the fair throughput of the technique, which can be complemented, when needed, by additional analyses by other surface-sensitive analytical techniques.
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| 7. |
- González-Domínguez, Álvaro, et al.
(författare)
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Untargeted Metabolomics Based on Liquid Chromatography-Mass Spectrometry for the Analysis of Plasma and Erythrocyte Samples in Childhood Obesity
- 2023
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; , s. 115-122
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The circulating metabolome of human peripheral blood provides valuable information to investigate the molecular mechanisms underlying the development of diseases and to discover candidate biomarkers. In particular, erythrocytes have been proposed as potential systemic indicators of the metabolic and redox status of the organism. To accomplish wide-coverage metabolomics analysis, the combination of complementary analytical techniques is necessary to manage the physicochemical complexity of the human metabolome. Herein, we describe an untargeted metabolomics method to capture the plasmatic and erythroid metabolomes based on ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry, combining reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography. The method provides comprehensive metabolomics fingerprinting of plasma and erythrocyte samples, thereby enabling the elucidation of the distinctive metabolic disturbances behind childhood obesity and associated comorbidities, such as insulin resistance.
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| 8. |
- Horvath, Istvan, 1979, et al.
(författare)
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In vitro analysis of α-synuclein amyloid formation and cross-reactivity
- 2018
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; , s. 73-83
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- In vitro time-resolved characterization of protein aggregation into amyloid fibers and the effects of other proteins on the aggregation process are fundamentally important measurements to obtain a better understanding of the mechanisms contributing to neurodegeneration, as well as other diseases involving amyloid formation. Here, we describe how to perform in vitro aggregation experiments with α-synuclein, the amyloidogenic protein involved in Parkinson’s disease, including how to assess the starting material, useful experimental/instrumental conditions, as well as how to set up cross-seeding and co-aggregation experiments. The high variability of data reported for in vitro α-synuclein amyloid formation may in part be explained by experimental differences.
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| 9. |
- Huang, Mingtao, 1984, et al.
(författare)
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High-throughput microfluidics for the screening of yeast libraries
- 2018
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Ingår i: Synthetic Metabolic Pathways. - New York, NY : Humana Press. - 9781493972944 ; , s. 307-317, s. 307-317
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Bokkapitel (refereegranskat)abstract
- Cell factory development is critically important for efficient biological production of chemicals, biofuels, and pharmaceuticals. Many rounds of the Design–Build–Test–Learn cycles may be required before an engineered strain meeting specific metrics required for industrial application. The bioindustry prefer products in secreted form (secreted products or extracellular metabolites) as it can lower the cost of downstream processing, reduce metabolic burden to cell hosts, and allow necessary modification on the final products, such as biopharmaceuticals. Yet, products in secreted form result in the disconnection of phenotype from genotype, which may have limited throughput in the Test step for identification of desired variants from large libraries of mutant strains. In droplet microfluidic screening, single cells are encapsulated in individual droplet and enable high-throughput processing and sorting of single cells or clones. Encapsulation in droplets allows this technology to overcome the throughput limitations present in traditional methods for screening by extracellular phenotypes. In this chapter, we describe a protocol/guideline for high-throughput droplet microfluidics screening of yeast libraries for higher protein secretion. This protocol can be adapted to screening by a range of other extracellular products from yeast or other hosts.
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| 10. |
- Humphrey, Madeleine, et al.
(författare)
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Tracking Global and Local Changes in Membrane Fluidity Through Fluorescence Spectroscopy and Microscopy
- 2023
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; , s. 203-229
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Membrane fluidity is a critical parameter of cellular membranes, which cells continuously strive to maintain within a viable range. Interference with the correct membrane fluidity state can strongly inhibit cell function. Triggered changes in membrane fluidity and associated impacts on lipid domains have been postulated to contribute to the mechanism of action of membrane targeting antimicrobials, but the corresponding analyses have been hampered by the absence of readily available analytical tools. Here, we expand upon the protocols outlined in the first edition of this book, providing further and alternative protocols that can be used to measure changes in membrane fluidity. We provide detailed protocols, which allow straightforward in vivo and in vitro measurement of antibiotic compound-triggered changes in membrane fluidity and fluid membrane microdomains. Furthermore, we summarize useful strains constructed by us and others to characterize and confirm lipid specificity of membrane antimicrobials directly in vivo.
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