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| 3. |
- Hauptmann, Giselbert, et al.
(author)
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Detection and signal amplification in zebrafish RNA FISH
- 2016
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In: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 98, s. 50-59
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Journal article (peer-reviewed)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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| 4. |
- Hober, Sophia, 1965-, et al.
(author)
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Bispecific applications of non-immunoglobulin scaffold binders
- 2019
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In: Methods. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1046-2023 .- 1095-9130. ; 154, s. 143-152
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Journal article (peer-reviewed)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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| 5. |
- Kaltashov, Igor A., et al.
(author)
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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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In: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 144, s. 14-26
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Research review (peer-reviewed)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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| 6. |
- Lamichhane, Santosh, et al.
(author)
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Gut metabolome meets microbiome : A methodological perspective to understand the relationship between host and microbe
- 2018
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In: Methods. - : Academic Press. - 1046-2023 .- 1095-9130. ; 149, s. 3-12
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Research review (peer-reviewed)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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| 7. |
- Lind, Christoffer, et al.
(author)
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Free energy calculations of RNA interactions
- 2019
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In: Methods. - : Elsevier. - 1046-2023 .- 1095-9130. ; 162-163, s. 85-95
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Journal article (peer-reviewed)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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| 9. |
- Shariatgorji, Mohammadreza, et al.
(author)
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Direct imaging of elemental distributions in tissue sections by laser ablation mass spectrometry
- 2016
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In: Methods. - : Elsevier BV. - 1046-2023 .- 1095-9130. ; 104, s. 86-92
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Journal article (peer-reviewed)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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| 10. |
- Tornmalm, Johan, et al.
(author)
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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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In: Methods. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1046-2023 .- 1095-9130. ; 140, s. 178-187
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Journal article (peer-reviewed)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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| 11. |
- Volkov, Ivan, et al.
(author)
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Tracking of single tRNAs for translation kinetics measurements in chloramphenicol treated bacteria
- 2019
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In: Methods. - : Elsevier. - 1046-2023 .- 1095-9130. ; 162-163, s. 23-30
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Journal article (peer-reviewed)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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| 12. |
- Wadsö, Lars
(author)
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A method for time-resolved calorespirometry of terrestrial samples.
- 2015
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In: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 76:Online 23 October 2014, s. 20-26
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Journal article (peer-reviewed)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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| 13. |
- Wadsö, Lars, et al.
(author)
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Calorespirometry of terrestrial organisms and ecosystems.
- 2015
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In: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 76:Online 28 October 2014, s. 11-19
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Journal article (peer-reviewed)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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| 14. |
- Wennmalm, Stefan, 1970-
(author)
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Potentials and pitfalls of inverse fluorescence correlation spectroscopy
- 2018
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In: Methods. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1046-2023 .- 1095-9130. ; 140, s. 23-31
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Journal article (peer-reviewed)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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| 16. |
- Sanzò, Gabriella, et al.
(author)
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A bimetallic nanocoral Au decorated with Pt nanoflowers (bio)sensor for H2O2 detection at low potential
- 2017
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In: Methods. - : Elsevier BV. - 1046-2023. ; 129, s. 89-95
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Journal article (peer-reviewed)abstract
- In this work, we have developed for the first time a method to make novel gold and platinum hybrid bimetallic nanostructures differing in shape and size. Au-Pt nanostructures were prepared by electrodeposition in two simple steps. The first step consists of the electrodeposition of nanocoral Au onto a gold substrate using hydrogen as a dynamic template in an ammonium chloride solution. After that, the Pt nanostructures were deposited onto the nanocoral Au organized in pores. Using Pt (II) and Pt (IV), we realized nanocoral Au decorated with Pt nanospheres and nanocoral Au decorated with Pt nanoflowers, respectively. The bimetallic nanostructures showed better capability to electrochemically oxidize hydrogen peroxide compared with nanocoral Au. Moreover, Au-Pt nanostructures were able to lower the potential of detection and a higher performance was obtained at a low applied potential. Then, glucose oxidase was immobilized onto the bimetallic Au-Pt nanostructure using cross-linking with glutaraldehyde. The biosensor was characterized by chronoamperometry at +0.15V vs. Ag pseudo-reference electrode (PRE) and showed good analytical performances with a linear range from 0.01 to 2.00mM and a sensitivity of 33.66μA/mMcm2. The good value of Km app (2.28mM) demonstrates that the hybrid nanostructure is a favorable environment for the enzyme. Moreover, the low working potential can minimize the interference from ascorbic acid and uric acid as well as reducing power consumption to effect sensing. The simple procedure to realize this nanostructure and to immobilize enzymes, as well as the analytical performances of the resulting devices, encourage the use of this technology for the development of biosensors for clinical analysis.
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