| 1. |
- Andreasson, Ulf, 1968, et al.
(author)
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Multiplexing and multivariate analysis in neurodegeneration.
- 2012
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In: Methods (San Diego, Calif.). - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 56:4, s. 464-70
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Journal article (peer-reviewed)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.
(author)
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A strategy for identification of protein tyrosine phosphorylation
- 2012
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In: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 56:2, s. 275-283
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Journal article (peer-reviewed)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. |
- Eriksson, Jan
(author)
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Detection and correction of interference in SRM analysis
- 2013
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In: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 61, s. 299-303
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Journal article (peer-reviewed)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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| 5. |
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| 6. |
- Howat, William J, et al.
(author)
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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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In: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 70:1, s. 34-38
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Journal article (peer-reviewed)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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| 7. |
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| 8. |
- Nilsson, Ola B., et al.
(author)
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Mammalian-derived respiratory allergens - Implications for diagnosis and therapy of individuals allergic to furry animals
- 2014
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In: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 66:1, s. 86-95
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Journal article (peer-reviewed)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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| 9. |
- Norrman, Karin, et al.
(author)
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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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In: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 59:1, s. 59-70
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Journal article (peer-reviewed)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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| 10. |
- Ståhlberg, Anders, 1975, et al.
(author)
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RT-qPCR work-flow for single-cell data analysis.
- 2013
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In: Methods (San Diego, Calif.). - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 59:1, s. 80-88
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Journal article (peer-reviewed)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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| 11. |
- Ståhlberg, Anders, 1975, et al.
(author)
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Single-cell gene expression profiling using reverse transcription quantitative real-time PCR.
- 2010
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In: Methods (San Diego, Calif.). - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 50:4, s. 282-8
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Research review (peer-reviewed)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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| 12. |
- Wählby, Carolina, et al.
(author)
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High- and low-throughput scoring of fat mass and body fat distribution in C. elegans
- 2014
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In: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 68:3, s. 492-499
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Journal article (peer-reviewed)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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| 13. |
- Pieringer, Astrid, 1979
(author)
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A numerical investigation of curve squeal in the case of constant wheel/rail friction
- 2014
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In: Journal of Sound and Vibration. - : Elsevier BV. - 1095-8568 .- 0022-460X. ; 333:18, s. 4295-4313
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Journal article (peer-reviewed)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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