| 1. |
- 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. ; 991, s. 127-137
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Otto, Maximilian, 1991, et al.
(författare)
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Saccharomyces cerevisiae as a Heterologous Host for Natural Products
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; 2489, s. 333-367
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Cell factories can provide a sustainable supply of natural products with applications as pharmaceuticals, food-additives or biofuels. Besides being an important model organism for eukaryotic systems, Saccharomyces cerevisiae is used as a chassis for the heterologous production of natural products. Its success as a cell factory can be attributed to the vast knowledge accumulated over decades of research, its overall ease of engineering and its robustness. Many methods and toolkits have been developed by the yeast metabolic engineering community with the aim of simplifying and accelerating the engineering process.In this chapter, a range of methodologies are highlighted, which can be used to develop novel natural product cell factories or to improve titer, rate and yields of an existing cell factory with the goal of developing an industrially relevant strain. The addressed topics are applicable for different stages of a cell factory engineering project and include the choice of a natural product platform strain, expression cassette design for heterologous or native genes, basic and advanced genetic engineering strategies, and library screening methods using biosensors. The many engineering methods available and the examples of yeast cell factories underline the importance and future potential of this host for industrial production of natural products.
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| 3. |
- Pigot, Harry, et al.
(författare)
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Ex Vivo Working Porcine Heart Model
- 2024. - 2
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Ingår i: Experimental Models of Cardiovascular Diseases : Methods and Protocols - Methods and Protocols. - 1940-6029 .- 1064-3745. - 9781071638484 - 9781071638453 - 9781071638460 ; 2803, s. 87-107
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Ex vivo working porcine heart models allow for the study of a heart’s function and physiology outside the living organism. These models are particularly useful due to the anatomical and physiological similarities between porcine and human hearts, providing an experimental platform to investigate cardiac disease or assess donor heart viability for transplantation. This chapter presents an in-depth discussion of the model’s components, including the perfusate, preload, and afterload. We explore the challenges of emulating cardiac afterload and present a historical perspective on afterload modeling, discussing various methodologies and their respective limitations. An actively controlled afterload device is introduced to enhance the model’s ability to rapidly adjust pressure in the large arteries, thereby providing a more accurate and dynamic experimental model. Finally, we provide a comprehensive experimental protocol for the ex vivo working porcine heart model.
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| 4. |
- Zorrilla, Francisco, 1994, et al.
(författare)
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Reconstruction of Genome-Scale Metabolic Model for Hansenula polymorpha Using RAVEN
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; 2513, s. 271-290
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Genome-scale metabolic models (GEMs) provide a useful framework for modeling the metabolism of microorganisms. While the applications of GEMs are wide and far reaching, the reconstruction and continuous curation of such models can be perceived as a tedious and time-consuming task. Using RAVEN, a MATLAB-based toolbox designed to facilitate the reconstruction analysis of metabolic networks, this protocol practically demonstrates how researchers can create their own GEMs using a homology-based approach. To provide a complete example, a draft GEM for the industrially relevant yeast Hansenula polymorpha is reconstructed.
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| 5. |
- Dash-Wagh, Suvarna, et al.
(författare)
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PepFect6 Mediated SiRNA Delivery into Organotypic Cultures
- 2016
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Ingår i: SiRNA Delivery Methods. - TOTOWA : HUMANA PRESS INC. - 9781493931125 - 9781493931118 ; 1364, s. 27-35
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Bokkapitel (refereegranskat)abstract
- Gene silencing by small interfering RNA (SiRNA) is an attractive therapeutic approach for pathological disorders that targets a specific gene. However, its applications are limited, as naked RNA is rapidly degraded by RNases and is inadequately internalized by the target cells in the body. Several viral and non-viral vectors have been described to improve the delivery of SiRNAs both in cultured cells as well as in vivo. Increasing evidence suggests that cell-penetrating peptides (CPPs) are an efficient, non-cytotoxic tool for intracellular delivery of SiRNA. Recently, a new peptide, PepFect6 (PF6), based system has been described for efficient SiRNA delivery in various cell types. PF6 is an amphipathic stearyl-TP10 peptide carrying a pH titratable trifluoromethylquinoline moiety that facilitate endosomal release. PF6 forms stable non-covalent complexes with SiRNA. Upon internalization, the complexes rapidly escape the endosomal compartment, resulting in robust RNA interference (RNAi) responses. This chapter describes a protocol to use the PF6-nanoparticle technology for SiRNA delivery into organotypic cultures of the inner ear i.e., cochlea. We also highlight different critical points in the peptide/SiRNA complex preparation, transfection and in analyzing the efficacy of PF6-SiRNA associated RNAi response.
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| 6. |
- Galaev, Igor, et al.
(författare)
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Affinity processing of cell-containing feeds using monolithic macroporous hydrogels, cryogels.
- 2008
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 421, s. 247-255
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Tidskriftsartikel (refereegranskat)abstract
- Monolithic macroporous hydrogels, "cryogels," are produced by polymerization in a partially frozen state when the ice crystals perform as a porogen. Cryogels have a unique combination of properties: (i) large (10-100 microm) pores; (ii) minimal non-specific interactions due to the hydrophilic nature of the polymers; (iii) porosities exceeding 80-90%; (iv) good mechanical stability. These properties of cryogels allow for their application for direct capture of extracellularly expressed histidine-tagged protein from the fermentation broth and separation of different cell types.
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| 7. |
- Johansson, Björn, et al.
(författare)
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Multiple Gene Expression by Chromosomal Integration and CRE-loxP-Mediated Marker Recycling in Saccharomyces cerevisiae
- 2004
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Ingår i: Recombinant Gene Expression Reviews and Protocols ( Methods in Molecular Biology ; 267 ). - New Jersey : Humana Press. - 1940-6029 .- 1064-3745. - 9781588292629 - 1592597742 ; 267
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK vector over again in the same strain after curing of the pCRE3 vector. The 2µ-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 is easily cured by growth in nonselective medium without active counterselection. The screening for loss of the chromosomal zeocin marker, as well as curing of the pCRE3 vector, is done in one step, by scoring zeocin sensitivity. This can be done because the zeocin marker is present in both the pB3 PGK and pCRE3. The S. cerevisiae pentose phosphate pathway genes RK11, RPE1, TAL1, and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in simultaneous overexpression of the genes in the xylose-fermenting S. cerevisiae strain TMB3001.
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| 8. |
- Rådström, Peter, et al.
(författare)
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Pre-PCR processing of samples.
- 2003
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Ingår i: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 216
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Tidskriftsartikel (refereegranskat)
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| 9. |
- Udatha, Gupta, 1984, et al.
(författare)
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Targeted metabolic engineering guided by computational analysis of single-nucleotide polymorphisms (SNPs).
- 2013
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029 .- 1064-3745. - 9781627032988 ; 985, s. 409-428
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Tidskriftsartikel (refereegranskat)abstract
- The non-synonymous SNPs, the so-called non-silent SNPs, which are single-nucleotide variations in the coding regions that give "birth" to amino acid mutations, are often involved in the modulation of protein function. Understanding the effect of individual amino acid mutations on a protein/enzyme function or stability is useful for altering its properties for a wide variety of engineering studies. Since measuring the effects of amino acid mutations experimentally is a laborious process, a variety of computational methods have been discussed here that aid to extract direct genotype to phenotype information.
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| 10. |
- Altai, Mohamed, et al.
(författare)
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Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting.
- 2020
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1064-3745 .- 1940-6029. ; 2105, s. 283-304, s. 283-304
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are small engineered scaffold proteins suitable for in vivo tumor targeting. Radionuclide molecular imaging using directly radiolabelled affibody molecules provides excellent imaging. However, affibody molecules have a high renal reabsorption, which complicates their use for radionuclide therapy. The high renal reabsorption is a common problem for the use of engineered scaffold proteins for radionuclide therapy. Affibody-based PNA-mediated pretargeting reduces dramatically the absorbed dose to the kidneys and makes affibody-based radionuclide therapy possible. This methodology might, hopefully, solve the problem of high renal reabsorption for radionuclide therapy mediated by other engineered scaffold proteins.
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