| 1. |
- Andreasson, Ulf, 1968, et al.
(författare)
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Multiplexing and multivariate analysis in neurodegeneration.
- 2012
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Ingår i: Methods (San Diego, Calif.). - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 56:4, s. 464-70
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Tidskriftsartikel (refereegranskat)abstract
- Limited sample volume is often an obstacle in clinical research and one way to circumvent this is to use multiplex techniques where several different analytes are simultaneously measured. There is a multitude of different platforms that can be used for multiplexing and their uniqueness and similarities will be described. Multivariate analysis is a powerful tool for extracting information from multiplex data. An introduction to one such algorithm is presented followed by examples from the literature, in the field of neurodegeneration, where multiplex and multivariate methods have been used.
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| 2. |
- Bergström Lind, Sara, et al.
(författare)
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A strategy for identification of protein tyrosine phosphorylation
- 2012
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 56:2, s. 275-283
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Tidskriftsartikel (refereegranskat)abstract
- To develop methods for studying phosphorylation of protein tyrosine residues is an important task since this protein modification regulates many cellular functions and often is involved in oncogenesis. An optimal protocol includes enrichment of tyrosine phosphorylated (pTyr) peptides or proteins, followed by a high resolving analytical method for identification of the enriched components. In this Methods paper, we describe a working strategy on how immunoaffinity enrichments, using anti-pTyr antibodies, combined with mass spectrometric (MS) analysis can be used to study the pTyr proteome. We describe in detail how our procedure was used to characterize the pTyr proteome of K562 leukemia cells. Important questions concerning the use of different anti-pTyr antibodies, enrichments performed at the peptide and/or the protein level, pooling of enrichments and requirements for the MS characterization are discussed.
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| 3. |
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| 4. |
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| 5. |
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| 6. |
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| 7. |
- Degerman, Eva, et al.
(författare)
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Phosphorylation and activation of hormone-sensitive adipocyte phosphodiesterase type 3B
- 1998
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 14:1, s. 43-53
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Tidskriftsartikel (refereegranskat)abstract
- Phosphodiesterases (PDEs) include a large group of structurally related enzymes that belong to at least seven related gene families (PDEs 1-7) that differ in their primary structure, affinity for cAMP and cGMP, response to specific effectors, sensitivity to specific inhibitors, and regulatory mechanism. One characteristic of PDE3s involves their phosphorylation and activation in response to insulin as well as to agents that increase cAMP in adipocytes, hepatocytes, and platelets and in response to insulin-like growth factor 1 in pancreatic beta cells. In adipocytes, activation of the membrane-associated PDE3B is the major mechanism whereby insulin antagonizes catecholamine-induced lipolysis. PDE3B activation results in increased degradation of cAMP and, thereby, a lowering of the activity of cAMP-dependent protein kinase (PKA). The reduced activity of PKA leads to a net dephosphorylation and decreased activity of hormone-sensitive lipase and reduced hydrolysis of triglycerides. Activation of the rat adipocyte PDE3B by insulin is associated with phosphorylation of serine-302. The mechanism whereby insulin stimulation leads to phosphorylation/activation of PDE3B is only partly understood. In rat adipocytes, lipolytic hormones and other agents that increase cAMP, including isoproterenol, also induce rapid phosphorylation, presumably catalyzed by PKA, of serine-302 of PDE3B. The phosphorylation is associated with activation of the enzyme, most likely representing "feedback" regulation of cAMP, presumably allowing close coupling of the regulation of steady-state concentrations of both cAMP and PKA and, thereby, control of lipolysis. In the review we describe methods and strategies used in the authors' laboratories to study phosphorylation and activation of PDE3B in adipocytes and in vitro.
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| 8. |
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| 9. |
- Eriksson, Jan
(författare)
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Detection and correction of interference in SRM analysis
- 2013
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 61, s. 299-303
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Tidskriftsartikel (refereegranskat)abstract
- Selected Reaction Monitoring (SRM) is a method of choice for accurate quantitation of low-abundance proteins in complex backgrounds. This strategy is, however, sensitive to interference from other components in the sample that have the same precursor and fragment masses as the monitored transitions. We present here an approach to detect interference by using the expected relative intensity of SRM transitions. We also designed an algorithm to automatically detect the linear range of calibration curves. These approaches were applied to the experimental data of Clinical Proteomic Tumor Analysis Consortium (CPTAC) Verification Work Group Study 7 and show that the corrected measurements provide more accurate quantitation than the uncorrected data. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.
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| 10. |
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| 11. |
- Guo, Xiaoxian, et al.
(författare)
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Understand the genomic diversity and evolution of fungal pathogen Candida glabrata by genome-wide analysis of genetic variations
- 2020
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 176, s. 82-90
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Candida glabrata, an opportunistic human fungal pathogen, is the second most prevalent cause of candidiasis worldwide, with an infection incidence that has been increasing in the past decades. The completion of the C. glabrata reference genome made fundamental contributions to the understanding of the molecular basis of its pathogenic phenotypes. However, knowledge of genome-wide genetic variations among C. glabrata strains is limited. In this study, we present a population genomic study of C. glabrata based on whole genome re-sequencing of 47 clinical strains to an average coverage of ∼63×. Abundant genetic variations were identified in these strains, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels) and copy number variations (CNVs). The observed patterns of variations revealed clear population structure of these strains. Using population genetic tests, we detected fast evolution of several genes involved in C. glabrata adherence ability, such as EPA9 and EPA10. We also located genome structural variations, including aneuploidies and large fragment CNVs, in regions that are functionally related to virulence. Subtelometric regions were hotspots of CNVs, which may contribute to variation in expression of adhesin genes that are important for virulence. We further conducted a genome-wide association study that identified two SNPs in the 5′UTR region of CST6 that were associated with fluconazole susceptibility. These observations provide convincing evidence for the highly dynamic nature of the C. glabrata genome with potential adaptive evolution to clinical environments, and offer valuable resources for investigating the mechanisms underlying drug resistance and virulence in this fungal pathogen. (249 words)
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| 12. |
- Hauptmann, Giselbert, et al.
(författare)
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Detection and signal amplification in zebrafish RNA FISH
- 2016
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 98, s. 50-59
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Tidskriftsartikel (refereegranskat)abstract
- In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts.
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| 13. |
- Hober, Sophia, 1965-, et al.
(författare)
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Bispecific applications of non-immunoglobulin scaffold binders
- 2019
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Ingår i: Methods. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1046-2023 .- 1095-9130. ; 154, s. 143-152
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Tidskriftsartikel (refereegranskat)abstract
- Non-immunoglobulin scaffolds represent a proven group of small affinity proteins that can be engineered in vitro to similar affinity and potency as monoclonal antibodies. Several novel candidate biotherapeutics that exploit the potential advantages scaffold proteins hold over larger and more complex antibodies have been developed over the past decade. The ease of using small and robust binding proteins as flexible and modular building blocks has led to the development of a wide range of innovative approaches to combine them in various bi- and multispecific formats. This progress is expected to aid the ongoing challenge of identifying niche applications where clear differentiation from antibody-based molecules will be key to success. Given the many engineering options that are available for non-immunoglobulin scaffold proteins, they have potential to not only complement but probably also surpass antibodies in certain applications.
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| 14. |
- Howat, William J, et al.
(författare)
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Antibody validation of immunohistochemistry for biomarker discovery : Recommendations of a consortium of academic and pharmaceutical based histopathology researchers
- 2014
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 70:1, s. 34-38
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Tidskriftsartikel (refereegranskat)abstract
- As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for "research use" continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1-3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier.
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| 15. |
- Kaltashov, Igor A., et al.
(författare)
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LC/MS at the whole protein level: Studies of biomolecular structure and interactions using native LC/MS and cross-path reactive chromatography (XP-RC) MS
- 2018
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 144, s. 14-26
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Forskningsöversikt (refereegranskat)abstract
- Interfacing liquid chromatography (LC) with electrospray ionization (ESI) to enable on-line MS detection had been initially implemented using reversed phase LC, which in the past three decades remained the default type of chromatography used for LC/MS and LC/MS/MS studies of protein structure. In contrast, the advantages of other types of LC as front-ends for ESI MS, particularly those that allow biopolymer higher order structure to be preserved throughout the separation process, enjoyed relatively little appreciation until recently. However, the past few years witnessed a dramatic surge of interest in the so-called “native” (with “non-denaturing” being perhaps a more appropriate adjective) LC/MS and LC/MS/MS analyses within the bioanalytical and biophysical communities. This review focuses on recent advances in this field, with an emphasis on size exclusion and ion exchange chromatography as front-end platforms for protein characterization by LC/MS. Also discussed are the benefits provided by the integration of chemical reactions in the native LC/MS analyses, including both ion chemistry in the gas phase (e.g., limited charge reduction for characterization of highly heterogeneous biopolymers) and solution-phase reactions (using the recently introduced technique cross-path reactive chromatography).
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| 16. |
- Kanno, T, et al.
(författare)
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Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements
- 2004
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 33:4, s. 302-311
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Tidskriftsartikel (refereegranskat)abstract
- This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic P-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other. (C) 2004 Elsevier Inc. All rights reserved.
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| 17. |
- Klepsch, Mirjam, 1983-, et al.
(författare)
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Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes
- 2008
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 46:2, s. 48-53
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Tidskriftsartikel (refereegranskat)abstract
- In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS–PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS–PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS–PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS–PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS–PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.
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| 18. |
- Kong, Xiangrui, et al.
(författare)
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Ordinal Analysis Applied to the Results of Positive Matrix Factorization of Chemical Ionization Mass Spectrometry Data
- 2020
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Ingår i: MethodsX. - : Elsevier BV. - 1046-2023 .- 1095-9130 .- 2215-0161. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- As an innovative analytical approach ordinal analysis is applied to positive matrix factorization (PMF) analysis outputs to identify the most important species and factors in chemical ionization mass spectrometry (CIMS) data. The procedure and outcome of the ordinal analysis facilitate further automated data analysis. Prior to standard PMF analysis, CIMS data were normalized to assure equal comparisons and facilitate the analysis process. The ordinal analysis was applied to the Factor Profiles (FPs) results, where mass numbers m/z are ranked by their FP fractions. Such ranking seeks the most influential compounds leading each factor, and the top m/z can be further investigated, e.g. by peak assignments. Rank maps can be plotted based on the ordinal results where the FPs are converted into a different space, which can potentially be used for cluster analysis. The rank maps provide an additional method for factor identification, especially when time series or other forms of the dataset are difficult to recognize. • Ordinal analysis identifies the most important fingerprint species leading each factor. • Rank map visualizes the features of each factors. • The method can be used as an online approach for source appointments of atmospheric pollutants.
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| 19. |
- Lamichhane, Santosh, et al.
(författare)
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Gut metabolome meets microbiome : A methodological perspective to understand the relationship between host and microbe
- 2018
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Ingår i: Methods. - : Academic Press. - 1046-2023 .- 1095-9130. ; 149, s. 3-12
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Forskningsöversikt (refereegranskat)abstract
- It is well established that gut microbes and their metabolic products regulate host metabolism. The interactions between the host and its gut microbiota are highly dynamic and complex. In this review we present and discuss the metabolomic strategies to study the gut microbial ecosystem. We highlight the metabolic profiling approaches to study faecal samples aimed at deciphering the metabolic product derived from gut microbiota. We also discuss how metabolomics data can be integrated with metagenomics data derived from gut microbiota and how such approaches may lead to better understanding of the microbial functions. Finally, the emerging approaches of genome-scale metabolic modelling to study microbial co-metabolism and host-microbe interactions are highlighted.
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| 20. |
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| 21. |
- Lind, Christoffer, et al.
(författare)
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Free energy calculations of RNA interactions
- 2019
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Ingår i: Methods. - : Elsevier. - 1046-2023 .- 1095-9130. ; 162-163, s. 85-95
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Tidskriftsartikel (refereegranskat)abstract
- This review discusses the use of molecular dynamics free energy calculations for characterizing RNA interactions, with particular emphasis on molecular recognition events involved in mRNA translation on the ribosome. The general methodology for efficient free energy calculations is outlined and our specific implementation for binding free energy changes due to base mutations in mRNA and tRNA is described, We show that there are a number of key problems related to the accuracy of protein synthesis that can be addressed with this type of computational approach and several such examples are discussed in detail. These include the decoding of mRNA during peptide chain elongation, initiation and termination of translation, as well as the energetic effects of base tautomerization and tRNA modifications. It is shown that free energy calculations can be made sufficiently reliable to allow quantitative conclusions to be drawn regarding the energetics of cognate versus non-cognate interactions and its structural origins.
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| 22. |
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| 23. |
- Mohammadi, M Rezaa, et al.
(författare)
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Isolation and characterization of microvesicles from mesenchymal stem cells.
- 2020
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 177, s. 50-57
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some extent. Microvesicles (MVs), also called as microparticles or ectosome, are among secreted vesicles that could transfer cytoplasmic cargo, including RNA and proteins, from emitting (source) cells to recipient cells. Given the importance of MVs, we here attempted to establish a method to isolate and characterize MVs secreted from unmodified human bone marrow derived MSCs (referred to as native MSCs, and their microvesicles as Native-MVs) and IFNγ stimulated MSCs (referred to as IFNγ-MSCs, and their microvesicles as IFNγ-MVs). We first describe an ultracentrifugation technique to isolate MVs from the conditioned cell culture media of MSCs. Next, we describe characterization and quality control steps to analyze the protein and RNA content of MVs. Finally, we examined the potential of MVs to exert immunomodulatory effects through induction of regulatory T cells (Tregs). Secretory vesicles from MSCs are promising alternatives for cell therapy with applications in drug delivery, regenerative medicine, and immunotherapy.
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| 24. |
- Nilsson, Ola B., et al.
(författare)
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Mammalian-derived respiratory allergens - Implications for diagnosis and therapy of individuals allergic to furry animals
- 2014
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 66:1, s. 86-95
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Tidskriftsartikel (refereegranskat)abstract
- Furry animals cause respiratory allergies in a significant proportion of the population. A majority of all mammalian allergens are spread as airborne particles, and several have been detected in environments where furry animals are not normally kept. The repertoire of allergens from each source belongs to a restricted number of allergen families. Classification of allergen families is particularly important for the characterization of allergenicity and cross-reactivity of allergens. In fact, major mammalian allergens are taken from only three protein families, i.e. the secretoglobin, lipocalin and kallikrein families. In particular, the lipocalin superfamily harbours major allergens in all important mammalian allergen sources, and cross-reactivity between lipocalin allergens may explain cross-species sensitization between mammals. The identification of single allergen components is of importance to improve diagnosis and therapy of allergic patients using component-resolved diagnostics and allergen-specific immunotherapy (ASIT) respectively. Major disadvantages with crude allergen extracts for these applications emphasize the benefits of careful characterization of individual allergens. Furthermore, detailed knowledge of the characteristics of an allergen is crucial to formulate attenuated allergy vaccines, e.g. hypoallergens. The diverse repertoires of individual allergens from different mammalian species influence the diagnostic potential and clinical efficacy of ASIT to furry animals. As such, detailed knowledge of individual allergens is essential for adequate clinical evaluation. This review compiles current knowledge of the allergen families of mammalian species, and discusses how this information may be used for improved diagnosis and therapy of individuals allergic to mammals.
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| 25. |
- Norrman, Karin, et al.
(författare)
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Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level.
- 2013
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 59:1, s. 59-70
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Tidskriftsartikel (refereegranskat)abstract
- Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE formation. Final DE differentiation was also analyzed with immunocytochemistry and single-cell gene expression profiling. We found that cells treated with activin A in combination with sodium butyrate and B27 serum-free supplement medium generated the most mature DE cells. Cell population studies were useful to monitor the temporal expression of genes involved in primitive streak formation and endoderm formation, while single-cell analysis allowed us to study cell culture heterogeneity and fingerprint individual cells. In addition, single-cell analysis revealed distinct gene expression patterns for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize cell types and subpopulations.
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| 26. |
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| 27. |
- Persson, Camilla, et al.
(författare)
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An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases
- 2005
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 35:1, s. 37-43
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Tidskriftsartikel (refereegranskat)abstract
- Regulation of protein tyrosine phosphatases (PTPs) through reversible oxidation of the active site cysteine is emerging as a general, yet poorly characterized, mechanism for control of the activity of this important group of enzymes. This regulatory mechanism was initially described after in vitro treatment of PTPs with oxidizing agents. However, accumulating evidence has substantiated the notion that this mechanism is also operating in vivo, e.g., in association with the transient increase in H(2)O(2) production which occurs after activation of receptor tyrosine kinases. A novel generic antibody-based method for monitoring of PTP oxidation is described. The sensitivity of this strategy has been validated by the demonstration of oxidation of endogenously expressed PTPs after stimulation of cells with growth factors. The method was also instrumental in providing the first evidence for intrinsic differences between PTP domains with regard to sensitivity to oxidation.
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| 28. |
- Selfhout, Maarten, et al.
(författare)
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Different types of Internet use, depression, and social anxiety : the role of perceived friendship quality
- 2009
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Ingår i: Journal of Adolescence. - : Wiley. - 0140-1971 .- 1095-9254. ; 32:4, s. 819-833
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Tidskriftsartikel (refereegranskat)abstract
- The current study examined the longitudinal associations of time spent on Internet activities for communication purposes (i.e., IM-ing) versus time spent oil Internet activities for non-communication purposes (i.e., surfing) with depression and social anxiety, as well as the moderating role of perceived friendship quality in these associations. Questionnaire data were gathered from 307 Dutch middle adolescents (average age 15 years) on two waves with a one-year interval. For adolescents who perceive low friendship quality, Internet use for communication purposes predicted less depression, whereas Internet use for non-communication purposes predicted more depression and more social anxiety. These results support social compensation effects of IM-ing on depression and poor-get-poorer effects of surfing on depression and social anxiety, respectively.
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| 29. |
- Shariatgorji, Mohammadreza, et al.
(författare)
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Direct imaging of elemental distributions in tissue sections by laser ablation mass spectrometry
- 2016
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 104, s. 86-92
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Tidskriftsartikel (refereegranskat)abstract
- We present a strategy for imaging of elements in biological tissues using laser ablation (LA) mass spectrometry (MS), which was compared to laser ablation inductively coupled plasma (LA-ICP) MS. Both methods were adopted for quantitative imaging of elements in mouse kidney, as well as traumatic brain injury model tissue sections. MS imaging (MSI) employing LA provides quantitative data by comparing signal abundances of sodium from tissues to those obtained by imaging quantitation calibration standards of the target element applied to adjacent control tissue sections. LA-ICP MSI provided quantitative data for several essential elements in both brain and kidney tissue sections using a dried-droplet approach. Both methods were used to image a rat model of traumatic brain injury, revealing accumulations of sodium and calcium in the impact area and its peripheral regions. LA MSI is shown to be a viable option for quantitative imaging of specific elements in biological tissue sections.
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| 30. |
- Shashkova, Sviatlana, 1987, et al.
(författare)
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Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells
- 2021
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 193, s. 46-53
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins play key roles at the interface between the cell and its environment by mediating selective import and export of molecules via plasma membrane channels. Despite a multitude of studies on transmembrane channels, understanding of their dynamics directly within living systems is limited. To address this, we correlated molecular scale information from living cells with real time changes to their microenvironment. We employed super-resolved millisecond fluorescence microscopy with a single-molecule sensitivity, to track labelled molecules of interest in real time. We use as example the aquaglyceroporin Fps1 in the yeast Saccharomyces cerevisiae to dissect and correlate its stoichiometry and molecular turnover kinetics with various extracellular conditions. We show that Fps1 resides in multi tetrameric clusters while hyperosmotic and oxidative stress conditions cause Fps1 reorganization. Moreover, we demonstrate that rapid exposure to hydrogen peroxide causes Fps1 degradation. In this way we shed new light on aspects of architecture and dynamics of glycerol-permeable plasma membrane channels.
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| 31. |
- Sjöbring, Ulf, et al.
(författare)
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Analysis of plasminogen-binding M proteins of Streptococcus pyogenes.
- 2000
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Ingår i: Methods. - 1095-9130. ; 21:2, s. 143-150
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Tidskriftsartikel (refereegranskat)abstract
- Group A streptococci are common human pathogens that cause a variety of infections. They express M proteins which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express M proteins that bind plasminogen and plasmin with high affinity. The binding is mediated by a 13-amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these M proteins. This sequence binds to kringle 2 in plasminogen, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of plasminogen. It could be demonstrated that plasminogen, absorbed from plasma by growing group A streptococci expressing the plasminogen-binding M proteins, could be activated by exogenous and endogenous streptokinase, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host.
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| 32. |
- Ståhlberg, Anders, 1975, et al.
(författare)
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RT-qPCR work-flow for single-cell data analysis.
- 2013
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Ingår i: Methods (San Diego, Calif.). - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 59:1, s. 80-88
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Tidskriftsartikel (refereegranskat)abstract
- Individual cells represent the basic unit in tissues and organisms and are in many aspects unique in their properties. The introduction of new and sensitive techniques to study single-cells opens up new avenues to understand fundamental biological processes. Well established statistical tools and recommendations exist for gene expression data based on traditional cell population measurements. However, these workflows are not suitable, and some steps are even inappropriate, to apply on single-cell data. Here, we present a simple and practical workflow for preprocessing of single-cell data generated by reverse transcription quantitative real-time PCR. The approach is demonstrated on a data set based on profiling of 41 genes in 303 single-cells. For some pre-processing steps we present options and also recommendations. In particular, we demonstrate and discuss different strategies for handling missing data and scaling data for downstream multivariate analysis. The aim of this workflow is provide guide to the rapidly growing community studying single-cells by means of reverse transcription quantitative real-time PCR profiling.
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| 33. |
- Ståhlberg, Anders, 1975, et al.
(författare)
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Single-cell gene expression profiling using reverse transcription quantitative real-time PCR.
- 2010
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Ingår i: Methods (San Diego, Calif.). - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 50:4, s. 282-8
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Forskningsöversikt (refereegranskat)abstract
- Even in an apparently homogeneous population of cells there are considerable differences between individual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while the single-cell response might be binary and apparently irregular. The origin of this variability may be preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements of individual cells. Here, we describe a method to collect dispersed single cells either by glass capillaries or flow cytometry, followed by quantitative mRNA profiling using reverse transcription and real-time PCR. We present a single cell lysis protocol and optimized priming conditions for reverse transcription. The large cell-to-cell variability in single-cell gene expression measurements excludes it from standard data analysis. Correlation studies can be used to find common regulatory elements that are indistinguishable at the population level. Single-cell gene expression profiling has the potential to become common practice in many laboratories and a powerful research tool for deeper understanding of molecular mechanisms.
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| 34. |
- Söderberg, Ola, et al.
(författare)
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Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.
- 2008
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 45:3, s. 227-32
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Tidskriftsartikel (refereegranskat)abstract
- The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.
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| 35. |
- Tornmalm, Johan, et al.
(författare)
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Label-free monitoring of ambient oxygenation and redox conditions using the photodynamics of flavin compounds and transient state (TRAST) spectroscopy
- 2018
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Ingår i: Methods. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1046-2023 .- 1095-9130. ; 140, s. 178-187
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Tidskriftsartikel (refereegranskat)abstract
- Transient state (TRAST) monitoring can determine population dynamics of long-lived, dark transient states of fluorescent molecules, detecting only the average fluorescence intensity from a sample, when subject to different excitation pulse trains. Like Fluorescence Correlation Spectroscopy (FCS), TRAST unites the detection sensitivity of fluorescence with the environmental sensitivity of long-lived non-fluorescent states, but does not rely on detection of stochastic fluorescence fluctuations from individual molecules. Relaxed requirements on noise suppression, detection quantum yield and time-resolution of the instrument, as well as on fluorescence brightness of the molecules studied, make TRAST broadly applicable, opening also for investigations based on less bright, auto-fluorescent molecules. In this work, we applied TRAST to study the transient state population dynamics within the auto-fluorescent coenzymes flavin adenine dinucleotide (FAD) and flavin-mononucleotide (FMN). From the experimental TRAST data, we defined state models, and determined rate parameters for triplet state and redox transitions within FMN and FAD, stacking and un-stacking rates of external redox active quenching agents and by the adenine moiety of FAD itself. TRAST experiments were found to be well capable to resolve these transitions in FMN and FAD, and to track how the transitions are influenced by ambient oxygenation and redox conditions. This work demonstrates that TRAST provides a useful tool to follow local oxygenation and redox conditions via FMN and FAD fluorescence, and forms the basis for measurements on flavoproteins and of redox and metabolic conditions in more complex environments, such as in live cells.
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| 36. |
- Volkov, Ivan, et al.
(författare)
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Tracking of single tRNAs for translation kinetics measurements in chloramphenicol treated bacteria
- 2019
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Ingår i: Methods. - : Elsevier. - 1046-2023 .- 1095-9130. ; 162-163, s. 23-30
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Tidskriftsartikel (refereegranskat)abstract
- Chloramphenicol is a broad-spectrum antibiotic targeting the protein synthesis machinery by binding to the bacterial ribosome. Chloramphenicol has been considered a classic general inhibitor of translation, blocking the accommodation of aa-tRNA into the A site of the large ribosomal subunit. However, recent studies suggest that this proposed mechanism is a simplification and that the effect of chloramphenicol on mRNA translation is much more dynamic. By tracking single dye-labelled elongator and initiator tRNAs in Escherichia coli cells treated with chloramphenicol, we observe the direct effect of chloramphenicol on translation kinetics. We find clear indications of slow but significant mRNA translation on drug bound ribosomes.
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| 37. |
- Wadsö, Lars
(författare)
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A method for time-resolved calorespirometry of terrestrial samples.
- 2015
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 76:Online 23 October 2014, s. 20-26
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Tidskriftsartikel (refereegranskat)abstract
- A new vessel for simultaneous isothermal calorimetry and respirometry (calorespirometry) on terrestrial (non-aqueous) samples has been developed. All types of small (<1g) biological samples (insects, soil, leaves, fungi, etc.) can be studied. The respirometric measurements are made by opening and closing a valve to a vial inside the sample ampoule containing a carbon dioxide absorbent. Typically a 7h measurement results in seven measurements of heat production rate, oxygen consumption and carbon dioxide production, which can be used to evaluate how the metabolic activity in a sample changes over time. Results from three experiments on leaves, a cut vegetable, and mold are given. As uncertainties - especially in the carbon dioxide production - tend to be quite high, improvements to the technique are also discussed.
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| 38. |
- Wadsö, Lars, et al.
(författare)
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Calorespirometry of terrestrial organisms and ecosystems.
- 2015
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 76:Online 28 October 2014, s. 11-19
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Tidskriftsartikel (refereegranskat)abstract
- Calorespirometry is the simultaneous measurement of heat and gas exchange from biological systems. Such measurements can be used to assess fundamental properties of many different types of systems from small ecosystems to isolated tissues. Techniques for calorespirometric measurements on terrestrial (non-aquatic) samples are described. Methods and models for evaluation of carbon conversion efficiencies, growth rates, and responses to environmental variables from calorespirometric measurements are described. A realistic model of the system under study is essential in the evaluation. Calorespirometry allows testing of models for tissues, individual organisms, and ecosystems.
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| 39. |
- Wennmalm, Stefan, 1970-
(författare)
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Potentials and pitfalls of inverse fluorescence correlation spectroscopy
- 2018
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Ingår i: Methods. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1046-2023 .- 1095-9130. ; 140, s. 23-31
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Tidskriftsartikel (refereegranskat)abstract
- Inverse Fluorescence Correlation Spectroscopy (iFCS) is a variant of FCS where unlabeled particles in solution, or domains in membranes, displace their surrounding, signal-generating molecules and thereby generate fluctuations. iFCS has to date been applied to unlabeled as well as labeled particles and protein molecules, using fluorescence as well as Raman scattering as a signal source, in diffraction-limited detection volumes as well as in nano-wells, and on fixed surfaces as well as in lipid bilayers. This review describes these applications and discusses the potentials and pitfalls when using iFCS.
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| 40. |
- Wählby, Carolina, et al.
(författare)
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High- and low-throughput scoring of fat mass and body fat distribution in C. elegans
- 2014
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Ingår i: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 68:3, s. 492-499
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Tidskriftsartikel (refereegranskat)abstract
- Fat accumulation is a complex phenotype affected by factors such as neuroendocrine signaling, feeding, activity, and reproductive output. Accordingly, the most informative screens for genes and compounds affecting fat accumulation would be those carried out in whole living animals. Caenorhabditis elegans is a well-established and effective model organism, especially for biological processes that involve organ systems and multicellular interactions, such as metabolism. Every cell in the transparent body of C. elegans is visible under a light microscope. Consequently, an accessible and reliable method to visualize worm lipid-droplet fat depots would make C. elegans the only metazoan in which genes affecting not only fat mass but also body fat distribution could be assessed at a genome-wide scale. Here we present a radical improvement in oil red O worm staining together with high-throughput image-based phenotyping. The three-step sample preparation method is robust, formaldehyde-free, and inexpensive, and requires only 15 min of hands-on time to process a 96-well plate. Together with our free and user-friendly automated image analysis package, this method enables C. elegans sample preparation and phenotype scoring at a scale that is compatible with genome-wide screens. Thus we present a feasible approach to small-scale phenotyping and large-scale screening for genetic and/or chemical perturbations that lead to alterations in fat quantity and distribution in whole animals.
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| 41. |
- Pieringer, Astrid, 1979
(författare)
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A numerical investigation of curve squeal in the case of constant wheel/rail friction
- 2014
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Ingår i: Journal of Sound and Vibration. - : Elsevier BV. - 1095-8568 .- 0022-460X. ; 333:18, s. 4295-4313
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Tidskriftsartikel (refereegranskat)abstract
- Curve squeal is commonly attributed to self-excited vibrations of the railway wheel, which arise due to a large lateral creepage of the wheel tyre on the top of the rail during curving. The phenomenon involves stick/slip oscillations in the wheel/rail contact and is therefore strongly dependent on the prevailing friction conditions. The mechanism causing the instability is, however, still a subject of controversial discussion. Most authors introduce the negative slope of the friction characteristic as a source of the instability, while others have found that squeal can also occur in the case of constant friction due to the coupling between normal and tangential dynamics. As a contribution to this discussion, a detailed model for high-frequency wheel/rail interaction during curving is presented in this paper and evaluated in the case of constant friction. The interaction model is formulated in the time domain and includes the coupling between normal and tangential directions. Track and wheel are described as linear systems using pre-calculated impulse response functions that are derived from detailed finite element models. The nonlinear, non-steady state contact model is based on an influence function method for the elastic half-space. Real measured wheel and rail profiles are used. Numerical results from the interaction model confirm that stick/slip oscillations occur also in the case of constant friction. The choice of the lateral creepage, the value of the friction coefficient and the lateral contact position on the wheel tread are seen to have a strong influence on the occurrence and amplitude of the stick/slip oscillations. The results from the interaction model are in good qualitative agreement with previously published findings on curve squeal.
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