| 1. |
- Andreasson, Erik, et al.
(författare)
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Isolation of Apoplast
- 2017
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745 .- 1940-6029. ; 1511, s. 233-240
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Tidskriftsartikel (refereegranskat)abstract
- The apoplast can be described as the soluble fraction of the extracellular space of plant tissue, and it plays an important role in signaling, nutrient transport, and plant-pathogen interactions. In this protocol, we describe a method where leaves are infiltrated with phosphate buffer under vacuum. The apoplast can then be extracted by centrifugation and simultaneously collected in a protease inhibitor solution. Using this protocol, typically 3 mu g of apoplastic proteins can be obtained in a volume of 300 mu L from five potato leaflets, with minimal contamination by non-apoplastic proteins.
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| 2. |
- Bus, Magdalena M., et al.
(författare)
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Forensic Analysis of Mitochondrial and Autosomal Markers Using Pyrosequencing®.
- 2015
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745 .- 1940-6029. ; 1315, s. 379-396
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Tidskriftsartikel (refereegranskat)abstract
- Forensic casework analyses often face challenges, such as limited genetic material with or without fragmentation and damage. To compensate for low amounts and degradation, shorter amplicons are often applied in the analysis. Also, a change of markers might be necessary using mitochondrial instead of autosomal markers. In addition, forensic research often involves analysis of large number of samples for marker evaluation and population-database compilation. Therefore, a flexible, robust but also rapid method for the detection of variation is highly useful. Pyrosequencing(®) is a rapid, reliable, easy-to-use method for sequence analysis. The method is well suited for rapid forensic analysis of a few targets or analysis of a single target in many samples. It allows sequencing of very short amplicons, which facilitates analysis of degraded DNA. Here we present the use of Pyrosequencing, a robust method for sensitive forensic analysis of mitochondrial DNA, autosomal STRs, and Y-chromosome STRs and SNPs.
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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
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| 8. |
- Grönbladh, Alfhild, et al.
(författare)
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[(35)S]GTPγS autoradiography for studies of opioid receptor functionality
- 2015
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745 .- 1940-6029. ; 1230, s. 169-176
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Tidskriftsartikel (refereegranskat)abstract
- The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR) that are Gi-coupled which make them suitable for studying the receptor functionality. The [(35)S]GTPγS autoradiography assay is a good option that has the benefit of generating both anatomical and functional data in the area of interest. It is based on the first step of the signaling mechanism of GPCRs. When a ligand binds to the receptor GTP will replace GDP on the α-subunit of the G protein, leading to a dissociation of the βγ-subunit. These subunits will start a cascade of second messengers and subsequently a physiological response.
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| 9. |
- 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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| 10. |
- 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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